UC-NRLF 


EMI    MEfl 


AGRIC,  DEPT, 


BIOLOGY 

LIBRARY 

G 


LABORATORY    DIRECTIONS 


FOR 


BEGINNERS  IN  BACTERIOLOGY 


BY 


VERANUS  A.  MOORE 


LABORATORY  DIRECTIONS 


FOR 


BEGINNERS  IN  BACTERIOLOGY 


BY 


VERANUS  A.  MOORE,  B.S.,  M.D. 

Professor  of  Comparative  Pathology  and  Bacteriology  and  of  Meat  Inspection, 
N.  Y.  State  Veterinary  College,  Cornell  University,  Ithaca,  N.  Y. 


PUBLISHED   BY  THE   AUTHOR. 

PRESS   OF   ANDRUS    &    CHURCH, 
ITHACA,  N.  Y. 


Main  Lib. 
Agrie.  Dept 


tsss 


COPYRIGHT,  1898, 
BY  VERANUS  A.  MOORE. 


st      - ;  Y, 
i898. 


TWO  COPIES  RECEIVED. 


PREFATORY  NOTE. 


It  has  been  found  desirable  to  provide  the  student,  just  beginning 
the  study  of  bacteriology,  with  a  somewhat  detailed  outline  of  the 
work  to  be  done  at  each  laboratory  session.  The  selecting  of  the  par- 
ticular things  to  be  done  and  the  choosing  of  methods  to  be  followed 
are  difficult  tasks.  The  assigning  of  directions  for  doing  work  under 
assumed  conditions  must  necessarily  partake  of  the  empirical,  and 
often  fail.  It  is  evident,  however,  that  practical  bacteriology  must, 
if  successfully  taught,  be  cast  in  a  somewhat  definite  form  in  order 
that  the  student  may  come  to  a  knowledge  of  the  fundamental  princi- 
ples underlying  the  subject  in  its  twofold  capacity,  that  of  a  pure  sci- 
ence and  of  a  useful  art. 

These  outlines  are  intended  to  serve  simply  as  a  guide  through  an 
introductory  laboratory  course  preparatory  either  to  independent  re- 
search work,  or  to  form  the  basis  for  the  application  of  the  principles 
of  bacteriology  in  the  practice  of  human  or  of  comparative  medicine. 
They  aim  to  impart  a  technical  and  working  knowledge  of  certain  of 
the  more  essential  methods  and  to  develop  a  definite  knowledge  of  a 
few  important  species  of  bacteria.  During  the  past  year,  they  were 
furnished  the  students  in  mimeographed  sheets,  but  after  making  the 
changes  suggested  by  this  application  it  seems  desirable  to  put  them 
in  a  more  convenient  form.  In  adjusting  the  amount  of  work  for 
each  exercise  to  the  necessary  limitations  of  time  and  facilities,  I  am 
indebted  to  Mr.  Raymond  C.  Reed,  Instructor  in  this  Department,  for 
much  valuable  assistance.  I  wish  also  to  thank  Prof.  Charles  Wright 
Dodge  of  the  University  of  Rochester  for  helpful  suggestions.  Should 
these  outlines  fall  in  the  hands  of  other  teachers  or  workers  in  this 
subject,  criticisms  are  cordially  invited. 


V.  A.  M. 


272069 


CONTENTS. 

PARTIAL  LIST  OF  TEXT  AND  REFERENCE  BOOKS.— APPA- 
RATUS AND  MATERIAL.— LABORATORY  MAXIMS. 


EXERCISBS.  PAGE. 

I.     Cleaning  glassware n 

II.  Plugging  test  tubes  and  flasks  and  sterilizing  the 

glassware 13 

III.  The  preparation -of  bouillon 14 

IV.  The  preparation  of  agar  and  gelatin 17 

V.     Inoculating  tubes  of  bouillon,  agar  and  gelatin 20 

VI.     The  examination  of  cultures 22 

VII.  Making  and  staining  cover-glass  preparations  and 

formulae  for  staining  solutions 25 

VIII.     Making  plate  and  Esmarch  roll  cultures 29 

IX.  The  examination  of  plate  cultures  and  making  sub- 
cultures from  colonies 32 

X.     The  preparation  of  certain  special  media 34 

XI.  Inoculating  special  media  and  examining  cultures. _  37 

XII.     The  examination  of  cultures  on  special  media 38 

XIII.  The  examination  of  cultures  continued 40 

XIV.  The  classification  of  bacteria 41 

XV.     Identifying  genera  among  bacteria 43 

XVI.     Studying  the  morphology  of  bacteria 44 

XVII.     Studying  and  staining  spores 45 

XVIII.     Staining  the  flagella  on  motile  bacteria 46 

XIX.     Staining  tubercle  bacteria  (bacilli) 49 

XX.     Making  cultures  of  anaerobic  bacteria 51 

XXI.     Examination  of  anaerobic  bacteria 52 

XXII.     Study  of  the  gas  production  by  bacteria _  53 

XXIII.  Identifying  genera  of  bacteria  and  obtaining  pure 

cultures  from  colonies 55 

XXIV.  Cleaning  used  culture  tubes,  flasks,  and  Petri  dishes,  56 
XXV.     Preparation  of  glassware  for  culture  media 56 

XXVI.     Preparation  of  culture  media 57 

XXVII.     A  study  of  certain  pyogenic  bacteria 58 

XXVIII.     Pyogenic  bacteria  (continued)   58 


VI 


Contents. 


EXERCISES.  PAGE. 

XXIX.     Bacillus  coli  communis 59 

XXX.     Bacillus  coli  com mun is  (continued) 61 

XXXI.     Bacillus  cholerae  suis  and  Bacillus  typhosus 62 

XXXII.     Bacillus  cholerae  suis  and  Bacillus  typhosus  (con- 
tinued)   63 

XXXIII.  Bacillus  cholerae  suis  and  Bacillus  typhosus  (con- 

tinued)   64 

XXXIV.  Widal  serum  test 65 

XXXV.     Bacterium  (Bacillus]    septicaemiae  hemorrhagicae 

and  Micrococcus  lanceolatus 66 

XXXVI.     Bacterium  (Bacillus}  septicaemiae  hemorrhagicae 

and  Micrococcus  lanceolatus  (continued) 67 

XXXVII.     Bacterium   (Bacillus]  septicaemiae  hemorrhagicae 

and.  Micrococcus  lanceolatus  (continued) 68 

XXXVIII.     Bacterium  (Bacillus}  tuberculosis 69 

XXXIX.     Bacterium  (Bacillus}  mallei 71 

XL/.     Bacterium  {Bacillus}  mallei  (continued) 71 

XLJ.     Actinomycosis 72 

XL/II.     Bacterium  (Bacillus}  anthracis 72 

XL/HI.     Bacterium  (Bacillus}  anthracis  (continued) 73 

XLJV.    Bacterium  (Bacillus}  diptheriae 74 

XL/V.     Bacterium  (Bacillus}  diptheriae  (continued) 76 

XLVI.     Determining  the  species  of  bacteria 77 

XL, VII.     Isolating  and  identifying  bacteria  from  animal  tissues  77 

XL/ VIII.     Isolating  and  identifying  bacteria  from  animal  tis- 
sues (continued) 79 

XL/IX.     The  examination  of  sections  of  tissues  containing 

bacteria 79 

I/.     Bacteriological  examination  of  pus  and  exudates 80 

I/I.     A  bacteriological  examination  of  the  skin  for  Micro- 
coccus  (Staphylococcus]  epidermidis  albus 81 

L/II.  Determining  the  thermal  death  point  of  bacteria 82 

I/HI.  Determining  the  efficiency  of  disinfectants 83 

LyIV.  Testing  disinfectants  ( continued ) 84 

L/V.     Pasteurizing  and  sterlizing  milk 85 

I, VI.     The  quantitative  bacteriological  examination  of  water  86 

L/VII.     The  quantitative  examination  of  water  ( continued )  _  _  87 

LVIII.     The  qualitative  examination  of  water 88 

LJX.     The  qualitative  examination  of  water  (continued)  __  89 

L/X.     Examination  of  certain  bacteria  not  studied  in  the 

laboratory 89 


A  PARTIAL  LIST  OF  TEXT  AND  REFERENCE 
BOOKS. 


Principles  of  Bacteriology.     By  A.  C.  Abbott. 

A  Manual  of  Bacteriology.     By  Surg.-Gen.  George  M.  Sternberg. 

A  Text-book  of  Bacteriology.     By  Surg.-Gen.  George  M.  Sternberg. 

Text-book  upon  the  Pathogenic  Bacteria.     By  Joseph  M'Farland. 

A  Text-book  of  Bacteriology  and  Infective  Diseases.     By  Edward  M. 

Crookshank. 

Manual  of  Bacteriology.     By  Muir  and  Ritchie. 
Micro-organisms  and  Disease.     By  E.  Klein. 
Bacteria  and  their  Products.     By  Sims  Woodhead. 
Micro-organisms  of  Water.     By  P.  and  G.  C.  Frankland. 
Bacteriological  Diagnosis.     By  J.  Bisenberg. 
Die  Microorganismen.     By  C.  Fliigge. 
Grundniss  der  Bakterienkundie.     By  C.  Fraenkel. 
Die  Methoden  der  Bakterien-Forschung.     By  F.  Hiippe. 
Lehrbuch  der  bakteriologischen  Untersuchung  und  Diagnostik.     By 

L.  Heim. 

Bacteriologische  Diagnostik.     By  I/ehmann  und  Neumann. 
System  der  Bakterien.     By  W.  Migula. 
Mikrophotographischer  Atlas  der  Bakterienkunde.     By  Fraenkel  und 

Pfeiffer. 

Jahresb.  u.d.  Fortsch.  d.  path  Mikroorganismen.     By  Baumgarten. 
Precis  de  Microbie.     By  Thoinot  et  Masselin. 
Precis  de  Bacte'riologie  clinique.     By  Wurtz. 
Microbiologie  Ve'terinaire.     By  Mosselman  and  Lienaux. 
L,es  Bacte"ries.     By  Cornil  et  Babes. 

JOURNALS  AND   PERIODICALS  OF  SPECIAL  VALUE. 

Centralblatt  fair  Bakteriologie  u.  Parasitenkunde  u.  Infektiouskrank- 

heiten. 

Journal  of  Pathology  and  Bacteriology. 
Zeitschrift  fur  Hygiene  u.  infectiouskrankheit. 
Annales  de  1'Institut  Pasteur. 

Archives  de  Med.  Experimental  et  d'anat.  Pathologique. 
See  also  standard  Medical  and  Veterinary  journals. 


LABORATORY  MAXIMS. 

1.  See  that  the  working  table,  instruments,  and  all  pieces  of  appar- 
atus used  are  thoroughly  cleaned  at  the  close  of  each  exercise. 

2.  Unless  otherwise  directed  all  cultures,  other  than  those  in  gelatin, 
are  to  be  grown  in  the  incubator. 

3.  Gelatin  cultures  should  not  be  put  in  the  incubator. 

4.  In  opening  tubes  of  media  or  cultures,  always  flame  the  open  end 
of  the  tube  immediately  after  withdrawing  the  plug.     If  the  tubes 
have  been  standing  for  some  time  the  surface  of  the  plug  should  be 
flamed  before  drawing  it  out.     Never  allow  the  tube  end  of  the  plug 
to  touch,  while  out  of  the  tube,  any  article  by  which  it  could  become 
contaminated.     It  should  be  held  by  the  top  between  the  fingers. 

5.  In  every  case  where  a  platinum  wire  loop  or  needle  is  used  for 
making  cultures  or  withdrawing  media  it  should  be  carefully  heated 
in  a  gas  flame  both  immediately  before  and  after  using.     The  heated 
wire  must  be  allowed  to  cool  before  making  cultures. 

6.  If  by  accident,  a  drop  or  more  of  a  culture  should  be  spilled  upon 
the  table  or  floor,  pour  over  it  a  sufficient  quantity  of  a  disinfectant 
(corrosive  sublimate  solution  i-iooo,  or  a  5%  solution  of  carbolic  acid) 
to  completely  cover  the  affected  area.     After  this  has  acted  for  ten 
minutes  wipe  it  up  and  boil  or  burn  the  cotton  or  cloth.     If  any  of  the 
culture  should  drop  on  the  hands  or  clothing  a  disinfectant  should  be 
applied  immediately. 

7.  In  sterilizing  culture  media,  always  see  that  there  is  enough  water 
in  the  pan  of  the  steam  sterilizer  or  in  the  water  bath  before  lighting 
the  gas.     Do  not  put  the  media  in  a  sterilizer  and  leave  the  laboratory. 

8.  Always  disinfect,  by  boiling,   all  cultures  before   cleaning  the 
tubes  or  plates  containing  them. 

9.  At  the  beginning  of  each  laboratory  session  read  the  directions 
for  the  next  exercise  in  order  to  be  able  to  make  any  preliminary 
preparations  which  may  be  required. 


APPARATUS  AND   MATERIAL. 

A.  For  general  use.     This  includes  the  apparatus  and  chemicals  to 
be  used  in  common  by  all  students.     It  consists  of  pans  and  brushes 
for  cleaning  test  tubes  and  other  glass  ware,  meat  mincer  and  press, 
large  and  small  water  baths,  steam  sterilizers,  hot-air  sterilizers,  incu- 
bators,   thermometers,  thermostats,  gas  burners,   balances,   leveling 
tripods,  Wolrrhiigels'  or  other  apparatus  for  aids  in  counting  colonies, 
micrometers,  metric  rules,  burettes,  tripods,  funnels,  beakers,  pipettes, 
graduates,  glass  tubing  and  rods.     The  chemicals  necessary  for  carry- 
ing, on  the  work,   such  as  various  acids  and  alkalies,  disinfectants, 
alcohol,  aniline  dyes,  and  those  articles  needed  in  the  preparation  of 
culture   media,   such  as  salt,  peptone,    agar,    gelatin,    meat   extract, 
sugars,  litmus  and  other  indicators,  and  filter  paper.     Fresh  meat, 
eggs,  milk  and  potatoes  must  be  furnished  as  needed.     It  also  includes 
color  charts  and  the  more  important  books  of  reference. 

B.  For  individual  use.     These  are  various  appliances  used  by  each 
student  and  for  which  he  becomes  personally  responsible.     They  con- 
sist of  a  microscope  with  substage  condenser,  two  oculars  (i  and  2  in.) 
and  three  objectives  (f ,  \  and  y*  inch),  a  bottle  of  immersion  oil,  a 
tripod  magnifier,  75  small  test  tubes,  50  large  test  tubes,  12  fermenta- 
tion tubes,  18  Petri  dishes,  3  Erlenmeyer  flasks,  7  one  ounce  bottles  for 
reagents  and  stains,   7  pipettes  with  rubber  bulbs  to  fit  bottles,  I 
platinum-wire  loop,   i  platinum-wire  needle,  3  tin  cups  for  holding 
cultures,  3  tin  boxes  for  holding  test  tubes,  i  block  for  holding  reagent 
bottles,  i  glass  slide  with  ring  attached  for  hanging-drop  preparations, 
i  tin  tray  for  cover-glass  preparations,  4  solid  watch  glasses,  2  Stender 
dishes  for  used  slides  and  cover-glasses,   and  a  glass  box  for  clean 
cover-glasses. 

Each  working  table  is  provided  with  a  reserve-flame,  gas  burner 
(Bunsen),  glass  jars  for  waste,  and  stands  for  holding  culture  tubes. 
A  requisite  amount  of  absorbent  cotton,  lens  paper,  and  towels  are 
furnished. 

Each  student  is  to  provide  himself  with  a  box  of  slides  and  cover- 
glasses  ( %  inch  square  cover-glasses  preferred ;  they  must  be  between 
.12  and  .18  mm.  in  thickness),  gummed  labels  for  slides  and  tubes,  a 
Faber's  blue  pencil  for  marking  on  glass,  fine  forceps  for  handling 
cover-glasses,  and  paper  for  laboratory  notes  with  manilla  card  board 
covers. 


EXERCISE  I. 

CLEANING  GLASSWARE. 

§  i.  Explanatory  note.  It  is  necessary  that  the  glass- 
ware employed  should  be  thoroughly  cleaned  before  it  is 
used.  Several  special  methods  have  been  suggested  for  this 
purpose  but  the  one  frequently  employed  by  chemists  seems 
to  be  the  most  easily  handled  and  quite  as  efficient  for 
general  use  as  the  more  elaborate,  specialized  processes.  It 
consists  in  applying  the  chromic  acid  cleaning  mixture  after 
washing  the  tubes  and  flasks  with  water.  It  is  sometimes 
necessary  to  employ  other  methods  for  cover-glasses  which 
are  to  be  used  in  staining  bacteria  where  a  mordant  is  re- 
quired. But  one  of  these  special  methods  will  be  given 
here. 

§  2.  General  directions.  Clean  all  of  the  glassware, 
test  tubes,  fermentation  tubes,  flasks,  Petri  dishes  and  reagent 
bottles  assigned. 

Put  the  slides  and  cover-glasses  in  the  cleaning  mixture, 
they  can  be  rinsed  and  wiped  later. 

Careful  notes  should  be  taken  on  the  work  of  each  exer- 
cise. These  should  be  handed  to  the  instructor  for  examina- 
tion and  correction  as  often  as  every  two  weeks. 

(a}  Test  tubes.  Wash  these  carefully  with  soap  and 
water  using  the  test  tube  brush.  After  washing  stand  them 
in  a  glass  jar  (aquarium)  and  fill  them  to  within  about  2  cm. 
of  the  top  with  cleaning  mixture  (this  will  be  kept  in  stock 
in  the  laboratory).  After  it  has  acted  for  from  5  to  10 
minutes  (it  is  better  to  stand  longer)  pour  it  out  of  the  tubes 
into  the  bottle  originally  containing  it.  Rinse  the  tubes 
thoroughly  in  tap  water  until  all  color  disappears  and  then 
in  hot  water  and  drain  them,  using  individual  drainage 


12 


beard ^ f rJDttE  locker*.  After^they  are  dry,  wipe  the  outside  of 
the'fribes^witti  a'siightiy.  dampened  cloth. 

(&)  Fermentation  tubes.  Treat  these  in  the  same  manner 
as  the  test  tubes  excepting  in  the  use  of  the  brush  which 
must  be  omitted. 

(V)  Flasks.  Wash  the  flasks  thoroughly  with  soap  and 
water.  Then  fill  them  with  the  cleaning  mixture  and  allow 
it  to  act  for  at  least  10  minutes  after  which  it  can  be  poured 
back.  Rinse  the  flasks  thoroughly  in  the  same  manner  as 
the  test  tubes  and  drain  them.  When  dry  the  outside  should 
be  wiped  with  a  damp  cloth. 

(d}  Petri  dishes  and  reagent  bottles.  Thoroughly  wash 
the  Petri  dishes  and  reagent  bottles  in  hot  soap  suds  after 
which  rinse  them  separately  in  hot  water.  Wipe  the  Petri 
dishes  dry  with  a  cloth  and  drain  the  bottles.  The  clean- 
ing mixture  need  not  be  used. 

(e)  Slides  and  cover-glasses.  Drop  the  cover-glasses 
singly  into  a  glass  jar  containing  cleaning  mixture  and  al- 
low them  to  remain  there  for  24  hours  or  longer.  Pour  off 
the  cleaning  mixture  and  rinse  the  cover-glasses  until  all  of 
the  color  disappears,  then  cover  them  with  alcohol  until 
needed  when  they  can  be  wiped  with  a  soft  linen  cloth 
or  with  lens  paper.  Treat  the  slides  in  the  same  manner  as 
the  cover-glasses.  They  can  be  wiped  directly  out  of  the 
rinsing  water.  The  slides  can  be  cleaned  satisfactorily  by 
washing  them  in  a  strong,  hot  soap  suds,  rinse  in  water  and 
wipe. 

(/)  Cleaning  used  culture  apparatus.  Place  the  tubes, 
flasks,  or  Petri  dishes  containing  old  cultures  in  a  water 
bath,  cover  them  with  water  to  which  add  a  little  sal  soda 
(about  an  ounce  to  the  gallon  of  water)  and  boil  for  20 
minutes.  Pour  off  the  water  and  empty  the  tubes  after 
which  again  boil  them  for  5  minutes  in  clean  soap  and  water. 
Then  wash  and  treat  with  the  cleaning  mixture  the  same  as 
the  new  tubes. 


§  3-  Formula  for  chromic  aQift  cje^nin^f^i^tiije^  ipjs- 
solve  80  grams  of  potassium  dichi'c/mat^{K2Cr2'd7)  in  300 
c.c.  of  warm  water,  when  all  of  the  K2  Cr2  O7  is  dissolved 
and  the  solution  cooled  add  it  slowly,  with  constant  stirring, 
to  460  c.c.  concentrated  sulphuric  acid  with  constant  stirring, 
store  the  mixture  in  a  glass-stoppered  bottle. 

§  4.  A  method  for  cleaning  cover-glasses  for  flagella 
stain.  For  this  work  the  ordinary  method  of  cleaning 
cover-glasses  is  not  sufficient.  The  following  treatment  was 
recommended  to  me  by  Dr.  Erwin  F.  Smith.  After  being 
cleaned  by  the  ordinary  method  boil  the  cover-glasses  in  an 
agate  cup  or  glass  beaker  in  a  10%  solution  of  caustic  soda 
for  five  minutes.  After  cooling  rinse  the  cover-glasses 
thoroughly  in  distilled  water  after  which  place  them  in  a 
beaker  and  cover  with  a  i%  solution  of  hydrochloric  acid, 
heat  to  the  boiling  point,  and  allow  to  stand  for  several 
hours  (over  night  or  longer).  Then  pour  the  acid  off  and 
rinse  the  cover-glasses  several  times  in  distilled  water  and 
finally  in  alcohol.  Wipe  them  out  of  alcohol  as  they  are 
needed. 


EXERCISE  II. 

PLUGGING  THE  TUBES  AND  FLASKS  AND  STERILIZING  THE 

GLASSWARE. 
> 

§  5.  Explanatory  note.  After  the  tubes  and  flasks  are 
cleaned  they  must  be  plugged.  The  plugged  tubes  and 
flasks  and  Petri  dishes,  all  of  which  are  to  be  used  for  hold- 
ing culture  media  or  in  making  cultures,  must  be  sterilized 
before  they  can  be  used.  The  plugs  should  be  neatly  made 
and  of  the  proper  length  and  firmness.  Absorbent  cotton  is 
ordinarily  used  for  this  purpose,  although  common  cotton  is 
employed  in  some  laboratories.  Glassware  is  sterilized  by 
means  of  dry  heat,  i.  e.,  in  the  hot  air  sterilizer.  (See 
methods  for  sterilizing  apparatus  and  instruments  in  text- 
books. ) 


I4 

.  Plug  all  of  the  tubes  and  flasks 
with  absorbent  co'tt'o'h'aYia  sterilize  them,  together  with  the  Pe- 
tri  dishes,  in  the  hot  air  sterilizer.  After  they  are  sterilized 
store  them  in  the  locker  until  they  are  needed.  The  Petri 
dishes  must  not  be  opened  until  they  are  used. 

§  7.  Plugging  the  tubes  and  flasks.  The  rolls  of  ab- 
sorbent cotton  are  cut  in  short  segments  of  from  5  to  7  cm. 
in  length.  A  piece  of  sufficient  length  of  this  narrow  strip 
to  give  cotton  enough  for  the  plug  is  torn  off.  The  quantity 
varies,  of  course,  with  the  size  of  the  mouth  of  the  tube,  or 
flask,  but  a  little  experience  will  enable  one  to  estimate  the 
quantity  accurately.  The  torn  edges  of  the  cotton  are 
turned  in  and  it  is  rolled  up  to  form  a  firm  plug  which  should 
snugly  fit  the  neck  of  the  tube  or  flask.  It  should  be  in- 
serted into  the  tube  for  about  2  cm.  and  the  end  should  be 
nearly  flat  and  smooth.  The  projecting  part  should  be 
about  the  same  length  and  be  of  equal  firmness. 

§  8.  Sterilizing  glass  ware.  Place  all  of  the  tubes, 
flasks,  and  Petri  dishes  in  the  hot  air  sterilizer,  close  the 
door  tightly  and  light  the  gas.  Heat  the  air  in  the  sterilizer 
to  a  temperature  of  from  135  to  150°  C.  and  keep  it  there 
for  one  hour,  ( the  temperature  must  not  be  allowed  to  go 
above  150°  C.  )  Then  turn  the  gas  off  and  when  the  tem- 
perature of  the  air  in  the  sterilizer  goes  down  to  or  below 
45  °  C.  the  door  can  be  opened  and  the  apparatus  removed. 


EXERCISE  III. 

THE  PREPARATION   OF  BOUILLON. 

§  9.  Explanatory  note.  Bouillon  is  the  liquid  medium 
most  commonly  employed  in  cultivating  bacteria.  It  is  prac- 
tically a  beef  tea  containing  peptone.  There  are  several 
methods  recommended  for  making  it.  ( i )  it  may  be  made 
directly  from  simple  meat  infusion  or,  (2)  it  may  be  made 
from  meat  extract.  The  meat  infusion  is  prepared  either  by 


15 

allowing  finely  chopped  lean  meat  mixed  with  twice  its  quan- 
tity of  water  (2  c.c.  of  water  for  each  gram  of  meat)  to 
stand  in  a  cool  place  for  from  12  to  18  hours,  or,  the  mix- 
ture of  meat  and  water  may  be  heated  with  frequent  stirring 
at  a  temperature  of  65°  C.  for  a  short  time  (one  hour). 
Each  method  has  its  peculiar  advantages.  When  the  meat 
is  macerated  at  a  low  temperature  the  muscle  sugar  is  fre- 
quently lost.  When  it  is  made  by  heating  the  meat  the 
bouillon  usually  contains  a  trace  of  muscle  sugar.  When 
meat  extract  is  used  in  place  of  the  meat  infusion  the  bouil- 
lon does  not  seem  to  be  a  favorable  culture  fluid  for  certain 
bacteria.  In  making  bouillon  therefore  it  becomes  necessary 
to  determine  the  kind  (whether  from  meat  infusion  or  ex- 
tract) and  the  method  of  preparing  it  to  suit  the  conditions 
in  hand.  It  is  sometimes  desirable  in  bacteriological  investi- 
gations to  resort  to  all  of  these  methods.  For  the  routine 
work  in  the  laboratory,  bouillon  prepared  directly  from  the 
meat  by  macerating  it  at  a  high  temperature  (65°  C.)  is 
very  satisfactory. 

Bouillon  is  used  as  the  nutritive  base  in  preparing  agar 
and  gelatin.  On  this  account  the  large  quantities  are  stored 
in  flasks.  (For  other  methods  see  text-books.  Also  Jour- 
nal of  the  American  Public  Health  Asso.,  Jan.  1898,  p.  77.) 

§  10.  General  Directions.  Make  1000  c.  c.  of  bouillon 
and  distribute  it  as  follows : 

Put  7  c.  c.  in  each  of  10  small  sterile  test  tubes. 

Put  300  c.  c.  in  each  of  2  flasks  and  the  balance  in  a  small 
flask. 

Put  7  c.  c.  of  distilled  water  in  each  of  5  small  sterile  test 
tubes  and  sterilize  them  with  the  bouillon.  These  tubes 
should  be  steamed  or  boiled  for  10  minutes  on  each  of  the 
two  succeeding  days.  (They  are  to  be  used  subsequently 
in  place  of  bouillon  in  making  dilutions).  Label  the  tubes, 
' '  Sterile  distilled  water. ' ' 

Note.     All  media,  in  addition  to  that  made  by  the  student, 


i6 

required  to  carry  out  the  directions  for  the  fall  term  will  be 
furnished. 

§n.  The  preparation  of  bouillon.  Take  500  grams  of 
lean  beef,  remove  all  fat,  and  grind  it  in  a  sausage  machine 
or  have  it  minced  at  the  butcher  shop.  Place  the  minced 
meat  in  an  agate  iron  dish  and  add  1000  c.  c.  of  distilled  or 
boiled  water,  (2  c.  c.  for  each  gram  of  meat)  and  thoroughly 
stir  with  a  glass  rod.  Then  macerate  it  with  frequent  stir- 
ring in  a  water  bath  at  a  temperature  of  65°  C.  for  i  hour 
after  the  temperature  of  the  meat  and  water  reaches  that  of 
the  water  outside.  Remove  the  meat  by  straining  the  liquid 
through  a  piece  of  cheese  cloth.  For  this  a  meat  press  is  de- 
sirable. The  liquid  should  equal  in  quantity  the  amount  of 
water  used,  if  it  does  not,  add  distilled  or  boiling  water  to 
make  it  up  to  that  amount.  To  this  meat  infusion  add  % 
per  cent,  peptone  (  Witte's  )  and  ^  per  cent,  sodium  chlor- 
ide. Add  enough  of  a  i  %  solution  of  sodium  hydrate  to 
give  the  liquid  a  faintly  alkaline  reaction.  In  this  work  the 
alkalinity  can  be  determined  by  the  use  of  sensitive  litmus 
paper.  (For  neutralizing  culture  media  for  special  or  re- 
search work,  see  Jour.  Am.  Pub.  H.  A.,  Jan.  1898,  p.  73.) 
The  infusion  is  then  boiled  in  a  water  bath  for  ^  of  an  hour, 
and  allowed  to  cool.  When  cool  filter  it  through  ordi- 
nary filter  paper.  The  filtrate  should  be  perfectly  clear. 
The  color  will  vary  according  to  the  amount  of  blood  pig- 
ment in  the  meat  used.  After  filtering,  distribute  the  bouil- 
lon in  tubes  and  flasks.  See  above.  Stand  the  tubes  con- 
taining the  bouillon  in  a  wire  basket  for  sterilization.  Ster- 
ilize them  by  boiling  in  a  closed  water  bath  or  steaming  in 
the  Arnold's  steam  sterilizer  for  30  minutes,*  the  time  to  be 


*  The  customary  method  of  sterilizing  culture  media  is  to  steam  or 
boil  it  for  about  10  minutes  on  each  of  three  consecutive  days.  This 
was  found  very  troublesome  by  the  students  and  feeling  that  it  was 
not  necessary  a  long  series  of  test  experiments  were  made  by  Mr.  R. 
C.  Reed,  who  found  that  one  boiling  or  steaming  for  30  minutes  gave 


17 

computed  from  the  time  the  water  boils.  The  flasks  of 
bouillon  should  be  boiled  or  steamed  for  20  minutes  on  each 
of  the  two  succeeding  days.  When  they  have  cooled,  the 
outside  of  the  tubes  should  be  carefully  wiped  with  a  moist 
cloth  and  placed  in  the  incubator  until  the  next  laboratory 
day.  Then  carefully  examine  them  and  if  any  of  the  tubes 
are  contaminated,  that  is,  if  the  liquid  is  clouded  or  has  a 
membrane  on  the  surface,  they  must  be  rejected.  Label  all 
others  and  place  them  in  the  locker. 

§  12.  Labeling.  Stick  on  each  tube  of  media,  about  3  cm. 
from  the  top,  an  adhesive,  white  label  about  2  cm.  square. 
On  the  upper  lines  should  be  written  the  name  of  the  me- 
dium, and  the  date  of  its  preparation.  Thus,  Bouillon,  13- 
VII-97.  When  the  tube  is  used  the  name  of  the  organism 
or  material  with  which  it  is  inoculated,  together  with  the 
date  of  inoculation,  should  be  written  on  the  lower  lines. 
This  applies  to  all  media  and  cultures. 


EXERCISE  IV. 

THE  PREPARATION  OF  AGAR  AND  GELATIN. 

§  13.  Explanatory  note.  Of  the  solid  media  employed 
in  cultivating  bacteria,  agar  and  gelatin  are  most  commonly 
used.  They  depend  for  their  nutritive  properties  largely 
upon  the  bouillon  from  which  they  are  made,  the  agar  and 
gelatin  forming  simply  the  solidifying  elements.  The  strik- 
ing difference  between  the  two  is  that  the  gelatin  melts  at 
the  body  temperature  whereas  the  agar  is  not  liquefied  below 
the  boiling  point.  For  this  reason  gelatin  cannot  be  used  as 


just  as  good  results  as  the  customary  3  boilings.  As  the  media  is  not 
used  for  2  or  3  days  after  its  sterilization  and  during  which  time  it  is 
kept  in  an  incubator,  the  method  is  adopted,  not  that  it  saves  time  in 
preparing  the  media,  but  it  relieves  the  congestion  in  the  sterilizer  and 
appreciably  aids  the  student. 


i8 

a  solid  medium  for  cultivating  bacteria  at  a  high  (body) 
temperature.  There  are  several  processes  for  preparing 
these  media  but  the  addition  of  the  dry  agar  and  gelatin  to 
bouillon  (§11)  either  immediately  after  it  is  filtered  or 
later  after  it  has  been  sterilized  and  stored  in  flasks  seems  to 
be  the  most  convenient  procedure.  The  agar  itself  is  usually 
neutral  in  reaction  but  the  gelatin  often  has  a  decidedly  acid 
reaction.  This  necessitates  the  careful  testing  of  the  reac- 
tion of  the  two  media. 

§  14.  General  directions.  Prepare  300  c.c.  of  agar  and 
300  c.c.  of  gelatin,  i.  e.,  start  with  300  c.c.  of  bouillon  for 
each.  There  will  be  considerable  shrinkage  so  that  the 
quantities  of  media  will  be  appreciably  less  than  this  amount. 
Distribute  each  medium  as  follows  : 

Put  7  c.c.  in  each  of  10  small  sterile  test  tubes. 
Put  12  c.c.  in  each  of  12  large  sterile  test  tubes. 
Put  the  balance  in  a  small  sterile  flask. 

$15.  Nutrient  gelatin.  Take  a  flask  of  bouillon  contain- 
ing 300  c.c.  and  pour  it  into  a  small  agate  iron  dish  and  add 
30  grams  of  sheet  gelatin,  which  has  been  cut  into  small 
pieces,  heat  the  bouillon,  with  frequent  stirring,  in  a  water 
bath  until  the  gelatin  is  dissolved.  Allow  it  to  cool  to  a 
temperature  between  45°  and  50°  C.  and  then  add  the  white 
of  one  egg  and  mix  it  thoroughly  by  stirring  or  better  by 
pouring  the  gelatin  from  one  flask  or  beaker  to  another. 
After  the  egg  albumen  is  completely  dissolved  return  the 
liquid  gelatin  to  the  large  covered  water  bath  and  boil  until 
the  egg  albumen  is  coagulated.  This  takes  about  20  min- 
utes. It  is  now  ready  for  filtering  which  must  be  done  while 
the  gelatin  is  hot.  Filter  through  properly  folded  but 
ordinary  filter  paper,  first  moistened  with  boiling  water. 
(For  illustrations  and  directions  for  folding  filter  paper  see 
Abbott's  Prin.  of  Bact.  p.  82).  Distribute  the  filtrate  as 
directed.  In  pouring  the  gelatin  into  the  tube  use  a  small 


19 

beaker  or  graduate  and  see  that  the  gelatin  does  not  touch 
the  sides  of  the  upper  part  of  the  tube.  Stand  the  tubes  in 
a  wire  basket  and  sterilize  them  by  boiling  in  a  closed  water 
bath  or  by  steaming  in  the  Arnold's  steam  sterilizer  for  30 
minutes.  The  small  flasks  can  be  sterilized  in  the  same 
manner.  Place  tubes  and  small  flasks  in  the  incubator  and 
allow  them  to  remain  there  for  two  days.  If  the  gelatin  in 
any  of  the  tubes  becomes  cloudy  the  medium  in  those  tubes 
must  be  rejected.  Carefully  wipe  all  of  the  other  tubes  with 
a  moist  cloth,  label  and  place  them  in  the  locker  where  they 
can  be  kept  until  used. 

§  1 6.  Nutrient  agar.  Weigh  out  3  grams  of  agar  and  cut 
it  into  small  pieces  with  a  pair  of  scissors.  Put  the  finely 
cut  agar  into  an  agate  iron  dish  and  add  50  c.c.  of  distilled 
water  and  boil  it  over  a  gas  flame  with  constant  stirring,  to 
prevent  scorching,  until  the  agar  is  dissolved,  giving  a  thick 
homogeneous  pasty  substance.  Pour  300  c.c.  of  bouillon 
(§  10)  from  a  flask  into  an  agate  iron  cup  and  to  it  add  the 
dissolved  agar.  Place  the  dish  containing  the  mixed  agar 
and  bouillon  in  a  closed  water  bath  and  boil  for  20  minutes. 
Then  cool  it  to  a  temperature  between  45  and  50°  C.  and 
add  the  white  of  one  egg  and  thoroughly  mix  it  in  the 
liquid  agar.  This  is  easily  accomplished  by  pouring  it  from 
one  beaker  to  another.  When  the  egg  albumen  is  dissolved 
the  agar  is  returned  to  the  water  bath  and  boiled  vigorously 
until  the  white  of  the  egg  is  coagulated.  This  usually  takes 
about  20  minutes.  Filter  the  agar  immediately,  while  hot, 
through  ordinary  filter  paper  which  has  been  moistened  with 
boiling  water.  Distribute  the  filtrate  in  small  and  large 
tubes,  as  directed.  Sterilize,  label,  and  store  the  agar  in  the 
same  manner  as  the  gelatin. 


20 


EXERCISE  V. 

INOCULATING  TUBES  OF  BOUILLON,  AGAR  AND  GELATIN. 

§  17.  General  directions.  Inoculate  a  tube  of  bouillon, 
two  (one  inclined  the  other  not)  of  agar  and  one  of  gelatin 
from  a  culture  of  Bacillus  coli  communis  which  will  be 
furnished. 

Wipe  out  the  slides  and  transfer  the  cover-glasses  to  the 
alcohol  (§  2  e). 

Read  the  chapters  in  the  text-books  on  inoculating  media 
or  making  tube  cultures. 

§  18.  Inoculating  bouillon.  In  making  this  culture  care- 
fully remove  the  plug  from  the  tube  of  bouillon  by  first 
twisting  it  around  to  detach  any  adhesions  and  then  pull  it 
straight  out.  Pass  the  open  end  of  the  tube  quickly 
through  the  gas  flame.  The  plug,  which  has  meantime 
been  carefully  held,  is  partially  replaced  and  the  tube  re- 
turned to  its  stand.  Treat  the  tube  containing  the  culture 
(which  has  been  furnished)  in  the  same  manner.  Then 
place  the  two  tubes  side  by  side  between  the  thumb  and  fore 
finger  of  the  left  hand  and  grasp  them  about  the  middle  of 
the  upper  half.  Sterilize  the  platinum  loop  by  passing  it 
through  the  gas  flame,  care  being  taken  that  the  handle  is 
flamed  for  a  distance  of  at  least  15  cm.  Then  carefully 
remove  the  plugs  from  the  tubes  and  hold  them  between  the 
fingers  in  such  a  manner  that  the  tube  ends  will  not  touch 
anything  during  the  inoculation  process.  Insert  the  wire 
loop  carefully  into  the  culture  and  transfer  a  loopful  of  the 
culture  to  the  tube  of  bouillon  and  gently  rinse  it  from  the 
loop.  The  loop  is  then  withdrawn,  the  plugs  replaced  in 
their  respective  tubes  and  the  loop  flamed  and  put  aside. 
Label  the  freshly  inoculated  tube  with  the  name  of  the 
organism,  source  and  date.  Stand  it  in  a  tray  or  cup  and 
place  it  in  the  incubator.  This  should  be  kept  at  a  temper- 


21 

ature  between  35  and  37 °C.  The  organism  thus  transferred 
should  multiply  so  that  on  the  following  day  the  liquid  will 
be  cloudy.  This  is  a  bouillon  culture  of  B.  coli  communis. 

§19.  Inoculating  tubes  of  agar.  Ordinarily  the  agar  is 
inclined  before  it  is  inoculated.  In  this  case  it  is  spoken  of 
as  inclined  or  slant  agar.  Occasionally  the  agar  is  inocu- 
lated without  inclining  it.  Cultures  made  in  this  manner 
are  spoken  of  as  "stab"  or  "stick"  cultures,  (i)  In- 
clined or  slant  agar.  Stand  a  tube  of  agar  in  a  wire  basket 
in  a  water  bath  and  boil  it  until  the  agar  is  liquefied.  (To 
save  repeating  this  it  is  well  to  incline  the  agar  in  several 
tubes  which  can  be  kept  for  future  use).  Lay  the  tubes  on 
a  tray,  the  top  resting  on  the  side  of  the  tray  so  that  the 
surface  of  the  agar  will  be  about  4  cm.  long,  allow  it  to  cool. 
In  placing  the  tubes  the  label  should  be  up.  When  the 
agar  has  set  it  is  ready  for  use.  It  is  inoculated  precisely  as 
the  bouillon  excepting  the  loopful  of  culture  is  drawn  over 
the  inclined  surface  instead  of  being  thrust  into  the  medium 
as  in  the  bouillon.  Label  and  place  it  in  the  incubator  with 
the  inoculated  bouillon  tube.  On  the  following  day  there 
should  be  a  grayish  growth  on  the  surface  of  the  agar  covered 
by  the  loop.  This  is  an  agar  culture  of  B.  coli  communis. 
(2)  Stick  cultures.  These  are  made  with  a  platinum  needle 
in  the  uninclined  agar.  The  impregnated  needle  is  pushed 
down  through  the  center  of  the  agar.  In  all  other  respects 
this  culture  is  made  like  the  slant  agar  culture. 

§  20.  Inoculating  the  tube  of  gelatin.  Tube  cultures  in 
gelatin  are  usually  made  without  inclining  the  gelatin,  i.e. , 
stick  cultures.  The  tube  of  gelatin  is  inoculated  in  the 
same  manner  as  the  stick  culture  in  agar.  This  tube  is  to 
be  placed  in  the  locker  as  the  gelatin  will  melt  at  the  incu- 
bator temperature.  The  growth  will  appear  in  about  two 
days  along  the  needle  track.  This  is  a  gelatin  culture  of 
B.  coli  communis. 


22 

EXERCISE  VI. 

THE  EXAMINATION  OF  CULTURES. 

§21.  Explanatory  note.  In  studying  cultures  of  bac- 
teria, it  is  necessary  to  observe  very  carefully  (i)  the  mac- 
roscopic appearance  of  the  growth  in  or  upon  the  media, 

(2)  the  microscopic  appearance  of  the  bacteria  in   (a)   the 
living  condition  (hanging-drop  preparation),  and  (b)  in  the 
dead   and  stained    condition  (cover-glass  preparation),  and 

(3)  the  effect  of  the  growth  of  the  bacteria  upon  the  chemi- 
cal  and  the   physical    properties  of   the  medium.     To  de- 
termine these,  the  cultures  must  be  kept  under  observation 
for  several  days  and  often  weeks.     A  careful  record  should 
be  made  of  the  changes  observed  in-  the  appearance  of  the 
cultures.     Illustrate  with  drawings. 

§22.  General  directions.  Examine  carefully  and 
describe  fully  the  appearance  of  the  bouillon,  agar,  and 
gelatin  cultures  made  in  Exercise  V. 

Determine  the  reaction  of  the  bouillon  culture  and  note 
whether  there  is  any  change  in  its  consistence  (viscidity). 

Make  a  hanging  drop  preparation  from  each  culture  and 
examine  and  describe  the  appearance  of  the  bacteria  in  each. 

Make  a  drawing  of  the  gelatin  and  slant  agar  cultures  and 
also  of  a  few  of  the  bacteria  in  one  of  the  hanging-drop  prep- 
arations. 

Read  the  paragraphs  in  the  text  books  on  the  examina- 
tion of  cultures  and  hanging  drop  preparations. 

§  23.  Suggestions  for  the  macroscopic  examination. 
The  external  appearance  of  cultures  should  be  observed  and 
noted  on  the  day  after  they  are  made  and  on  each  succeed- 
ing day  until  the  growth  ceases.  In  bouillon  cultures  the 
appearance  of  the  liquid,  whether  uniformly,  faintly  or 
heavily  clouded,  turbid,  clear  or  clouded  with  flocculent 
masses  held  in  suspension,  the  quantity,  and  nature  of  sedi- 


23 

ment,  and  the  presence  or  absence  of  a  membrane  should  be 
noted.  The  reaction  of  the  liquid,  its  consistence  and  odor 
should  be  determined.  In  the  agar  cultures  the  extent  of 
the  growth  (feeble,  moderate  or  vigorous,)  its  color,  form 
and  surface  appearance  (dull  or  glistening)  should  be  ob- 
served. The  character  of  the  growth  in  the  condensation 
water  should  also  be  noted.  In  stab  cultures  the  appearance 
of  the  growth  both  on  the  surface  and  along  the  needle  track 
should  be  described.  In  gelatin,  the  absence  or  the  presence 
and  extent  of  liquefaction  should  be  noted  in  addition  to  the 
features  already  referred  to  for  the  stab  agar  cultures. 

§  24.  Testing  the  reaction  of  liquid  cultures.  Place  a 
small  piece  of  each  of  the  red  and  blue  litmus  paper  in  a 
solid  watch  glass.  With  the  platinum  loop  carefully  place  a 
drop  of  the  culture  on  each  piece  of  the  paper.  After  re- 
cording the  reaction  produced,  neutral,  acid  or  alkaline  with 
the  degree,  cover  the  paper  with  a  disinfectant  (a  solution 
of  corrosive  sublimate  i  to  1000).  After  it  has  acted  for 
about  10  minutes  empty  it  with  the  paper  into  the  waste  jar 
and  wash  the  watch  glass. 

To  determine  the  viscidity.  (i)  Bouillon  cultures. 
Insert  the  platinum  loop  into  the  liquid  and  carefully  with- 
draw it.  The  approximate  degree  of  viscidity  can  be  de- 
termined by  the  extent  of  the  adhesion  of  the  liquid  to  the 
loop,  and  the  length  of  the  thread-like  filament  drawn  out. 
By  gently  shaking  the  tube  a  viscid  sediment  will  rise  up 
appearing  as  a  somewhat  twisted  tenacious  cone  with  its 
apex  reaching  to  or  near  the  surface.  A  friable  sediment 
will  break  up  and  become  disseminated  through  the  liquid 
upon  agitation.  (2)  Agar  and  gelatin  cultures.  Touch  the 
surface  growth  with  the  end  of  the  platinum  needle  and  if 
it  is  viscid  a  thread-like  string  will  be  drawn  out.  Note 
whether  the  growth  is  pasty  or  friable. 

§  25.  Making  hanging-drop  preparations,  (i)  From 
bouillon  culture.  Place  a  clean  cover-glass  on  the  tray. 


24 

With  the  loop,  remove  a  drop  of  the  liquid  culture  and 
place  it  on  the  middle  of  the  cover-glass.  With  a  pair  of 
fine  forceps  invert  the  cover-glass  over  the  glass  ring  fixed 
to  a  slide  for  this  purpose.  The  surface  of  the  ring  should 
previously  be  moistened  with  liquid  vaseline  to  prevent  the 
cover-glass  from  sliding.  The  preparation  is  then  ready  for 
examination.  Examine  it  first  with  the  high  power  dry  lens 
and  then  with  the  oil  immersion  objective.  (For  directions 
in  the  use  of  the  microscope  see  "  The  Microscope"  by  Pro- 
fessor S.  H.  Gage.)  (2)  From  cultures  on  solid  media.  On 
account  of  the  very  large  number  of  bacteria  in  the  growth 
on  solid  media  it  is  necessary  to  separate  them  in  a  clear 
liquid.  Take  a  cover-glass  as  before  and  place  a  loopful  of 
sterilized  water  or  bouillon  on  the  center.  With  the  plati- 
num needle  touch  the  surface  growth  very  gently  with  the 
end  of  the  needle  and  carefully  rinse  it  in  the  drop  of  liquid 
on  the  cover-glass.  From  this  point  the  examination  is  the 
same  as  with  the  liquid  culture.  Upon  examination,  if  the 
bacteria  are  so  numerous  that  the  individual  organisms  can 
not  be  clearly  distinguished,  i.e.,  separated  from  each  other, 
the  preparation  must  be  rejected  and  another  one  made, 
using  a  smaller  quantity  of  the  growth. 

§  26.  Suggestions  for  the  microscopic  examination  of 
bacteria.  In  examining  the  bacteria,  as  they  appear  under 
the  microscope  in  the  hanging  drop  preparations,  the  follow- 
ing features  should  be  observed  :  Are  the  individual  bacteria 
spherical,  rod-shaped  or  spiral  in  form?  Are  they  single  or 
united  in  pairs,  masses  or  clumps,  or  in  shorter  or  longer 
chains  ?  For  this  determination  it  is  better  to  examine  the 
edge  of  the  drop.  Are  they  motile,  that  is,  do  the  individual 
bacteria  move  from  one  point  in  the  field  to  another  ?  To 
determine  this  the  center  of  the  drop  is  better.  Clearly  dis- 
tinguish between  motility  and  a  simple  dancing  motion. 
Determine  the  presence  or  absence  of  spores.  These  are 
bright  highly  refractory  bodies  either  within  or  outside  of 


25 

the  bodies  of  the  bacteria.  If  present  they  can  usually  be 
seen  in  both  positions.  Is  there  any  evidence  of  a  capsule 
around  the  bacteria  ? 


EXERCISE  VII. 

MAKING    AND     STAINING    COVER  GLASS    PREPARATIONS, 
AND  FORMULAE  FOR  STAINING  SOLUTIONS. 

§  27.  General  Directions.  Make  two  cover-glass  prepa- 
rations from  each  of  the  cultures  made  in  Exercise  V  and  stain 
one  with  alkaline  methylene-blue  and  the  other  with  carbol 
fuchsin.  Describe  the  appearance  of  the  bacteria  and  make 
a  drawing  of  a  few  individual  bacteria  from  the  preparations 
made  from  the  agar  culture. 

Preserve  a  cover-glass  preparation  mounted  in  balsam  and 
labeled  to  accompany  notes. 

Prepare  the  staining  fluids  used  in  this  exercise  from  the 
formulae  given. 

Read  the  paragraphs  in  the  text-books  on  making  and 
staining  cover-glass  preparations. 

§  28.  Making  cover-glass  preparations,  (i)  From 
bouillon  cultures.  Place  2  clean  cover-glasses  on  the  tray. 
With  the  loop  remove  a  drop  of  the  bouillon  culture  and 
spread  it  in  a  thin  layer  over  about  y$  of  the  surface  of  the 
cover-glasses.  One  loopful  will  ordinarily  make  from  2  to  4 
preparations.  Allow  the  liquid  to  dry  on  the  cover-glasses 
in  the  air.  When  dry  fix  the  bacteria  to  the  cover-glasses 
by  passing  them,  film  upward,  3  times  through  the  middle 
of  the  upper  half  of  the  gas  flame.  Each  passage  (complete 
circle)  should  not  occupy  more  than  one  second.  After  fix- 
ing they  are  ready  for  staining  (§  29).  (2)  From  cultures 
on  solid  media  (agar,  gelatin,  potato,  serum,  etc.).  Place 
the  cover-glasses  on  the  tray  and  on  the  center  of  each  a 


26 

drop  of  sterile  water  or  bouillon.  With  the  needle  touch 
the  surface  growth  of  the  culture  and  then  gently  rinse  the 
end  of  the  needle  in  the  liquid  on  the  covers.  Spread  the 
liquid  on  the  covers  as  before.  From  this  point  the  procedure 
is  the  same  as  that  for  the  preparations  made  from  the  bouil- 
lon culture. 

§  29.  Staining  bacteria  in  cover-glass  preparations. 
(i)  With  alkaline  methjdene-blue.  With  the  pipette  place 
a  few  drops  of  the  staining  solution  on  the  film  side  of  the 
preparation  which  is  either  held  horizontally  with  the  fine 
forceps  or  left  resting  on  the  tray.  Allow  the  stain  to  act 
for  2  or  3  minutes.  Then  carefully  rinse  off  the  stain  in 
water,  holding  the  cover  firmly  by  one  edge  with  the  for- 
ceps. After  thoroughly  rinsing  place  the  preparation,  film 
downward,  on  a  clean  slide  and  dry  the  upper  surface  with  a 
piece  of  filter  paper.  It  is  now  ready  for  the  microscopic 
examination.  Use  first  the  dry  lens  (^j  in.  obj.)  and  then 
the  oil  immersion  objective.  If  the  specimen  is  a  good  one 
and  it  is  desirable  to  preserve  it  wipe  off  the  drop  of  oil  with 
apiece  of  lens  paper,  run  a  drop  of  distilled  water  under  the 
cover-glass  which  floats  it,  when  it  can  be  easily  removed 
with  the  forceps.  Place  it  on  the  tray,  film  upward,  and 
when  dry  mount  it  in  alkaline  Canada  balsam.  (2)  With 
carbol-fuchsin.  Cover  the  film  on  the  cover-glass  with  the 
stain  and  allow  it  to  act  for  about  one  minute.  Then  rinse 
it  thoroughly  in  water  after  which  cover  it  with  -£$%  so- 
lution of  acetic  acid  or  strong  (95  %)  alcohol.  Allow  this  to 
act  from  5  to  10  seconds  and  again  thoroughly  rinse  in  water 
and  examine  as  above.  (For  other  decolorizers,  see  text- 
books. ) 

Upon  examination  the  preparation  should  be  free  from 
deposits  or  stained  back  ground.  The  bacteria  should,  as  a 
rule,  be  isolated  and  distinct,  unless  the}7  are,  the  prepara- 
tions are  not  satisfactory. 

Cover-glass    preparations   of    bacteria   are    permanently 


27 

mounted  in  the  same  manner  as  similar  preparations  made 
from  the  blood  or  other  tissues  in  histology,  the  process  be- 
ing to  place  a  drop  of  the  balsam  on  the  center  of  the  slide 
and  place  the  preparation,  film  downward,  over  it  and  apply 
slight  pressure.  Isabel  the  preparation,  giving  the  name  of 
the  organism,  its  source,  (kind  of  culture,  tissue,  etc.,  from 
which  the  preparation  was  made),  stain  used,  and  date.  If 
the  specimen  is  not  preserved  the  slide  and  cover-glass 
should  be  cleaned  for  future  use. 

§  30.  Suggestions  concerning  the  microscopic  exam- 
ination of  stained  preparations  of  bacteria.  In  the  ex- 
amination of  the  bacteria  in  the  stained  condition  the  fol- 
lowing points,  and  perhaps,  others  should  be  observed  and 
noted,  (i)  Concerning  their  morphology.  Are  they  spher- 
ical, rod-shaped,  or  spiral?  Are  they  separated  or  united  in 
clumps  or  chains?  If  rod-shaped,  are  the  ends  pointed, 
round,  or  square  ?  Are  the  bacteria  all  of  the  same  form  and 
.size  ?  Note  the  presence  or  absence  of  spores  and  capsules. 
(2)  Concerning  their  reaction  to  staining  fluids.  Do  they 
stain  uniformly  or  irregularly?  Do  they  stain  deeply  or 
faintly  ?  Is  the  center  lighter  than  the  periphery  ?  Is  there 
an  unstained  central  band  and  deeply  stained  ends  (polar 
stain)  ?  Do  all  of  the  bacteria  take  the  stain  alike? 

§  31.  Staining  solutions.  The  basic  aniline  dyes  are 
used  in  staining  bacteria.  There  is  a  large  number  of  these, 
and  there  are  several  formulae  for  preparing  staining  solu- 
tions from  each.  Further,  as  will  be  seen  from  the  chapters 
on  staining  bacteria  in  the  text-books,  there  are  several 
methods  of  applying  these  stains.  In  an  introductory  course, 
however,  it  is  impossible  to  try  them  all  and  consequently 
those  are  described  which  seem  to  be  the  best  adapted  for 
general  use. 

In  addition  to  the  ordinary  staining  solutions  and  methods 
there  are  special  processes  for  certain  species  such,  for 
example,  as  the  tubercle  bacillus,  and  still  others  for  stain- 


28 

ing  certain  parts  of  many  bacteria,  such  as  the  flagella  on 
motile  forms,  the  spores  in  spore  bearing  organisms,  and 
the  capsule  on  certain  other  species.  There  is  a  large  num- 
ber of  these  special  methods  but  in  this  course  only  one  of 
each  will  be  given.  These  will  be  taken  up  in  connection 
with  the  study  of  the  bacteria  requiring  them. 

§  32.  Formulae  for  staining  solutions.  The  dyes  used 
are  methylene-blue,  gentian-violet,  methyl-violet,  and 
fuchsin. 

I,OEFFI,ER'S  AI,KAUNE  METHYI,ENE-BUJE. 

Concentrated  alcoholic  solution  of  methylene-blue 30    c.c. 

Caustic  potash  i  per  cent,  solution i      c.c. 

Distilled  water 100  c.c. 

CARBOI,  FUCHSIN  (ZIEHI/S  SOLUTION). 

Fuchsin  (dry) i  gram. 

Alcohol    (Absolute) 10    c.c. 

Carbolic  acid,  5  per  cent,  solution 100  c.c. 

Dissolve  the  fuchsin  in  the  alcohol,  after  which  add  the 
carbolic  acid  solution.  Instead  of  using  the  dry  fuchsin 
and  alcohol,  10  c.c.  of  a  saturated  alcoholic  solution  of 
fuchsin  may  be  used. 

§  33.  Aqueous  Solutions.  Aqueous  solutions  of  methyl- 
violet,  gentian- violet,  fuchsin,  and  the  other  aniline  dyes 
are  prepared  by  adding  i  c.c.  of  the  saturated  alcoholic 
solution  of  the  desired  dye  to  20  c.c.  of  distilled  water. 
This  will  impart  a  decided  color  to  the  liquid  so  that  a 
pipette  full  will  be  barely  transparent. 

The  true  aqueous  solutions  are  made  by  dissolving  the  dyes 
in  water,  but  these  are  weak  and  not  so  effective  as  those 
prepared  from  the  alcoholic  solutions.  These  solutions 
deteriorate  in  a  short  time.  The  carbol  fuchsin  and  alkaline 
methylene-blue  will  keep  a  little  longer,  but  they  require  to 
be  filtered  occasionally. 


29 

ANILINE  METHYI,- VIOLET  (EHRUCH-WEIGERT). 

Saturated  alcoholic  solution  of  methyl-violet n  c.c. 

Absolute  alcohol 10  c.c. 

Aniline  water 100  c.c. 

§  34.  Making  aniline  water.  Aniline  water  is  a  saturated 
aqueous  solution  of  aniline  oil.  It  is  prepared  by  adding  i 
c.c.  of  aniline  oil  to  20  c.c.  of  distilled  water  and  shaking 
frequenth''  for  from  15  to  30  minutes.  It  is  convenient  to 
use  a  stoppered  vial  or  large  test  tube  for  mixing  it.  Filter 
through  a  moistened  filter  paper.  The  filtrate  should  be 
perfectly  clear.  If  it  is  cloudy  it  should  be  refiltered  before 
using. 


EXERCISE  VIII. 

MAKING  PLATE  AND  ESMARCH  ROLL  CULTURES. 

§  35.  Explanatory  note.  The  general  principle  under- 
lying the  separation  of  bacteria  by  means  of  plate  and  roll 
cultures  is  to  dilute  the  substance  containing  the  bacteria  so 
that  the  individual  organisms  will  be  separated  from  each 
other  by  an  appreciable  distance  and  then  fixed  in  a  solid 
medium  where  each  organism  can  multiply  into  a  growth 
or  colony  without  coming  in  contact  with  any  other 
organism  or  colony.  For  this  purpose  agar  and  gelatin  are 
used.  Originally,  Koch  employed  a  rectangular  piece  of 
glass  for  holding  the  layer  of  medium  and  protected  it  from 
contamination  by  putting  it  under  a  bell  jar.  Later 
Esmarch  introduced  the  ' '  roll  culture ' '  method  which  was 
extensively  followed,  until  the  Petri  dishes  were  introduced. 
Since  that  time  they  have  been  largely  used  in  place  of  the 
Koch  plate  and  Esmarch  tube.  On  this  account  the  plate 
cultures  of  to-day  are  usually  made  in  Petri  dishes.  The 
roll  culture,  however,  is  occasionally  made. 


30 

§  36.  General  Directions.  Make  a  series  of  3  agar  plates, 
one  of  3  gelatin  plates,  and  a  series  of  3  gelatin  roll  cul- 
tures (Esmarch  rolls)  from  the  bouillon  culture  of  Bacillus  coli 
communis  (§  18).  Place  the  agar  plates  in  the  incubator  and 
the  gelatin  plates  and  rolls  in  a  locker  for  that  purpose. 

Re-examine  all  the  cultures  made  in  previous  exercises 
and  add  to  the  laboratory  notes  a  description  of  any  changes 
in  their  appearance.  The  notes  should  contain  a  detailed 
record  of  the  cultures  made  in  this  exercise. 

Read  carefully  the  paragraphs  in  the  text  books  on  mak- 
ing plate  and  roll  cultures. 

§37.  Making  agar  plates.  Take  three  large  tubes  of  agar, 
stand  them  in  a  water  bath  aild  boil  until  the  agar  is  lique- 
fied. Then  cool  by  standing  the  tubes  with  a  thermometer 
in  a  cup  of  water  at  a  temperature  of  about  50°  C.  As  the 
temperature  rises  add  a  little  cold  water.  When  the  temper- 
ature of  the  agar  reaches  that  of  the  water  and  the  temper- 
ature of  the  whole  has  lowered  to  45°  C.  the  agar  is  ready 
for  use.  For  convenience  in  labeling  number  the  tubes  i  f 
2,  and  3. 

Place  3  sterilized  Petri  dishes  on  the  leveling  tripod  and 
adjust  it  by  means  of  a  spirit  level.  With  the  wire  loop  pro- 
ceed by  the  same  method  as  followed  in  making  bouillon 
cultures.  Take  one  loopful  of  the  bouillon  culture  and  place 
it  in  agar  tube  No.  i  and  mix  by  carefully  shaking  it. 
Flame  the  wire  and  transfer  two  loopfuls  of  agar  from  tube 
i  to  tube  2  and  mix  as  before.  Again  flame  the  loop  and 
transfer  3  loopfuls  from  tube  2  to  tube  3  and  mix  as  with 
tubes  i  and  2.  After  the  tubes  are  inoculated,  pour  the 
agar  into  the  Petri  dishes.  In  doing  this  remove  the  plug, 
flame  the  mouth  of  the  tube,  and  after  quickly  cooling,  raise 
with  the  left  hand  the  edge  of  the  cover  on  one  side  of  -the 
dish  sufficiently  to  allow  of  inserting  the  mouth  of  the  tube, 
and  hold  it  until  the  agar  is  poured  and  replace  it  immedi- 
ately. Label,  and  number  the  Petri  dishes  to  correspond 


with  the  dilutions  in  the  tubes,  thus,  plate  i  is  from  tube 
one,  plate  2  is  from  tube  2,  and  plate  3  is  from  tube  3.  In 
making  the  dilutions  it  is  important  that  the  wire  loop  should 
be  flamed  after  making  each  transfer. 

§  38.  Making  gelatin  plates.  These  are  prepared  pre- 
cisely as  the  agar  plates  with  these  exceptions.  ( i )  The 
gelatin  is  liquefied  at  a  temperature  of  45°  C.  (2)  The 
plates  when  made  are  to  be  kept  in  the  locker  the  same  as 
the  gelatin  .stab  cultures.  (3)  In  hot  weather  it  is  some- 
times necessary  to  put  a  piece  of  ice  in  the  reservoir  under 
the  glass  plate  on  the  leveling  tripod  to  congeal  the  gelatin. 

The  directions  given  above  for  making  the  dilutions  are 
applicable  only  when  the  original  culture  is  moderately 
clouded.  If  there  are  comparatively  very  few  bacteria  in 
the  liquid  a  larger  quantity  of  the  culture  will  be  necessary. 
If  there  are  many  more,  as  in  turbid  bouillon  or  slant  agar 
culture,  it  will  be  necessary  to  take  a  much  smaller  quantity 
for  the  first  dilution.  It  is  often  desirable  to  make  the  first 
dilution  in  a  tube  of  sterile  water  or  bouillon  instead  of  gela- 
tin or  agar,  and  to  make  two  rather  than  three  plates. 

§39.  Making  Esmarch  roll  cultures.  For  this  purpose 
gelatin  is  ordinarily  used  as  agar  does  not  adhere  readily  to- 
the  sides  of  the  tubes.  It  is  sometimes  used.  Take  the  de- 
sired number  of  large  tubes  of  gelatin,  liquefy,  inoculate, 
label  and  number  the  dilutions  as  in  making  gelatin  plate 
cultures.  Place  a  block  of  ice  about  6  inches  long  in  an 
agate  iron  tray.  Melt  a  slight  horizontal  groove  in  the  ice 
with  a  test  tube  containing  hot  media  or  water.  The  inocu- 
lated tubes  are  tipped  and  rolled  so  that  the  liquid  gelatin 
moistens  the  inside  of  the  tube  to  within  about  a  centimeter 
of  the  plug.  Then  roll  the  tube  rapidly  in  the  groove  on 
the  ice  until  the  medium  becomes  solid.  The  gelatin  should 
not  come  in  contact  with  the  plug.  In  rolling  the  tube  the 
plugged  end  should  always  project  beyond  the  ice.  See 
illustration  in  text  books. 


EXERCISE  IX. 

THE    EXAMINATION    OF   PLATE   CULTURES  AND  MAKING 
SUBCULTURES  FROM  COLONIES. 

§  40.  Explanatory  note.  In  practical  bacteriological 
work  plate  cultures  are  made  use  of  in  determining  (i)  the 
number  of  bacteria  there  is  in  a  given  substance;  (2)  the 
different  species  of  bacteria  present,  and  (3)  the  character  of 
the  growth  in  a  colony  of  the  organism  in  question.  Other 
important  facts,  such,  for  example,  as  the  relative  number 
of  each  species  of  bacteria,  or  the  difference  in  the  appear- 
ance of  the  surface  and  deep  colonies  are  learned  through 
this  process.  The  plate  culture,  therefore,  is  one  of  the 
most  important  single  methods  employed  in  isolating  and 
studying  bacteria. 

§  41.  General  directions.  Examine  carefully  and 
describe  the  plate  cultures  made  in  Exercise  VIII.  If  the 
agar  plates  do  not  have  colonies,  or  if  the  colonies  are  so 
numerous  that  they  cannot  be  counted  on  any  of  the  plates, 
make  the  cultures  over  again,  and  give  an  explanation  in  the 
notes  of  this  exercise  for  the  failure  to  obtain  good  results. 

Make  a  hanging  drop  preparation  from  a  colony  from  an 
agar  plate,  and  one  from  a  colony  from  a  gelatin  plate,  and 
examine  them  microscopically.  Describe  the  appearance  of 
the  bacteria  in  each. 

Make  a  cover-glass  preparation  from  the  same  colonies 
and  stain  each  with  carbol  fuchsin.  Examine  each  carefully 
and  make  a  drawing  of  a  few  of  the  isolated  bacteria. 
Describe  (§30)  the  appearance  of  the  bacteria  in  these 
preparations. 

Inoculate  a  tube  of  bouillon,  one  of  agar  and  one  of  gela- 
tin from  a  well  isolated  colony  from  one  of  the  agar  plates. 

§  42.  Suggestions  for  the  examination  of  the  plate  and 
roll  cultures.  Observe  the  general  appearance  of  the 


33 

plates,  note  whether  the  colonies  are  well  isolated  or  run 
together  (confluent)  ;  describe  the  appearance  of  the  indi- 
vidual colonies,  (a)  those  on  the  surface,  (b)  those  in  the 
depth  of  the  medium.  Indicate  their  shape  (round,  lenticu- 
lar, flat,  convex  or  spherical).  Are  the  edges  sharply 
defined  ?  Is  the  margin  even  or  irregular  ?  Give  their  size 
(diameter  in  millimeters),  and  indicate  their  color  (deter- 
mine shade  from  a  color  chart),  and  consistence.  Do  the 
surface  colonies  adhere  to  the  medium  or  can  they  be  easily 
removed  ?  Examine  them  with  a  low  lens  and  describe  the 
surface  markings  if  any.  Also  indicate  the  difference  in 
color  as  observed  with  the  unaided  eye  and  with  the 
microscope. 

§  43.  Estimating  the  number  of  colonies  on  plates.  If 
the  number  of  colonies  is  not  large  (not  to  exceed  100)  they 
can  be  counted  and  the  exact  number  recorded.  This  may 
be  done  with  the  third  plate.  When  the  number  is  larger  it 
is  more  convenient  to  divide  the  total  area  into  smaller  ones 
and  count  the  number  of  colonies  in  each  of  several  (20  to 
40)  of  the  small  areas,  add  these  together  and  divide  the 
sum  by  the  number  of  areas  counted.  The  quotient  gives 
the  average  number  on  one  area ;  multiply  this  quotient  by 
the  number  of  areas  containing  colonies  and  the  product 
will  be  the  number  of  colonies  on  the  plate.  This  latter 
process,  however,  gives  the  approximate  number  only. 

For  dividing  the  area  of  the  plate  into  smaller,  equal  areas, 
it  is  convenient  to  use  Wolff hiigel's  counting  apparatus. 
This  was  devised  more  particularly  for  square  or  oblong 
plates  (Koch).  In  counting  the  colonies  on  the  Petri  dishes 
Parker's  scheme  modified  by  Jeffers  is  more  suitable.  It 
consists  of  a  disc  about  20  cm.  in  diameter  divided  into  areas 
of  a  square  centimeter  each.  Place  the  Petri  dish  over  the 
disc  taking  care  that  it  is  accurately  centered. 

Count  the  number  of  colonies  in  several  (20  to  40)  of  the 
areas  and  multiply  the  mean  number  by  the  number  of  areas 


34 

covered.     This  product  gives  the  approximate  number  of 
colonies. 

§  44.  Making  subcultures  from  colonies.  Select  the 
tubes  of  media  to  be  used,  and  flame  the  mouths  as  hereto- 
fore described.  Select  a  colony  as  well  isolated  from  all 
others  as  possible.  With  the  left  hand  carefully  raise  the 
edge  of  one  side  of  the  cover  of  the  Petri  dish,  and  while 
holding  it,  touch  the  colony  with  the  needle,  replace  the 
cover,  take  up  the  tube  of  media  and  inoculate  it.  If 
bouillon  is  used  first  a  tube  of  agar  or  gelatin  can  be  inocu- 
lated immediately  afterwards  without  recharging  the  needle. 
If  more  cultures  are  to  be  made  it  is  necessary  to  again 
charge  the  needle  from  the  colony.  If  the  plate  is  to  be  re- 
jected the  cover  can  be  entirely  removed  in  the  beginning. 
The  newly  inoculated  tubes  or  subcultures  should  be  labeled 
and  treated  according  to  the  directions  heretofore  given  for 
handling  cultures.  These  inoculated  tubes  should  be  pure 
cultures.  It  sometimes  happens,  however,  that  what  appears 
to  be  a  single  colony  consists  of  the  growth  of  two  organisms. 
If  these  should  be  of  different  species  the  cultures  made 
from  the  colony  would  probably  be  impure.  These  impure 
growths  (apparently  single  colonies)  frequently  develop  on 
plate  cultures  exposed  to  the  air  for  some  time.  The 
particles  of  dust  often  carry  two  or  more  bacteria. 


EXERCISE  X. 

THE  PREPARATION  OF  CERTAIN  SPECIAL  MEDIA. 

§  45.  Explanatory  note.  In  studying  the  properties  of 
bacteria  it  is  desirable  to  cultivate  them  on  a  number  of  dif- 
ferent media.  Bouillon,  agar,  and  gelatin  are  most  com- 
monly used,  but  others  are  necessary  in  determining  the 
cultural  peculiarities  and  important  biochemic  properties  of 
the  organism  in  question.  The  cultivation  of  bacteria  upon 


35 

these  media  may  be  regarded  somewhat  as  a  test,  to  deter- 
mine the  presence  or  absence  of  certain  properties.  Thus, 
for  example,  will  the  species  in  hand  coagulate  the  casein  in 
milk,  produce  gas  in  media  containing  saccharose,  grow  on 
potato,  etc.?  The  number  of  these  tests  which  have  been 
used  and  recognized  as  important  is  quite  large,  but  in  a 
short  course  only  those  possessed  of  the  more  differential 
value  can  be  tried.  In  describing  a  new  species,  or  identi- 
fying any  of  the  carefully  described  ones,  it  is  important  to 
know  at  least  some  of  these  cultural  peculiarities  and  bio- 
chemic  properties.  For  this  reason  it  is  necessary  to  learn 
the  method  of  preparation  and  the  use  of  certain  of  these 
media. 

In  addition  to  the  above,  a  few  species  of  bacteria  require 
a  particular  kind  or  kinds  of  media  for  their  diagnostic  or 
most  differential  growth. 

Among  these  are  those  of  glanders,  diphtheria  and  tuber- 
culosis. The  preparation  of  these  particular  media  will  be 
considered  in  connection  with  the  study  of  the  organisms 
requiring  them. 

§  46.  General  Directions.  Prepare  for  culture  media, 
5  tubes  of  potatoes,  5  tubes  of  milk,  5  tubes  of  litmus  milk, 
5  tubes  of  glucose  agar,  5  tubes  of  glycerin  agar,  4  fermenta- 
tion tubes  of  bouillon  containing  glucose,  4  containing 
lactose,  and  4  containing  saccharose.  (The  agar  and  the 
sugar  free  bouillon  necessary  in  the  work  of  this  exercise 
will  be  furnished  by  the  instructor.) 

Read  carefully  the  paragraphs  in  the  text  books  on  the 
preparation  and  use  of  these  media. 

§  47.  Preparation  of  potato  for  a  culture  medium. 
Select  medium  sized  potatoes,  thoroughly  wash  and  cut  out, 
with  a  cutter  made  for  this  purpose,  a  cylinder  3  to  4  cm. 
long  (oblong  pieces  cut  with  a  knife  will  do  quite  as  well). 
Ordinarily  2  cylinders  can  be  cut  from  each  potato.  The 
inclined  surface  is  obtained  by  cutting  out  the  potato  pro- 


36 

jecting  above  the  frame  of  the  cylindrical  knife.  All  of  the 
skin  must  be  removed.  Wash  the  potato  cylinders  in  cold, 
running  water  for  some  5  minutes  (a  longer  time  is  prefer- 
able) and  place  them  in  test  tubes  of  the  proper  size  (large 
or  small  according  to  size  of  cutter  used),  and  add  about  i 
c.c.  of  water  to  each  tube.  Sterilize  them  by  discontinuous 
boiling  or  steaming  for  20  minutes  each  day  for  three  con- 
secutive days.  Wipe,  label,  and  store  in  locker. 

§  48.  Preparation  of  milk  for  a  culture  medium. 
Place  about  100  c.c.  of  fresh  milk  in  a  beaker  in  the  ice  box 
and  allow  it  to  stand  for  from  10  to  15  hours.  Then  care- 
fully remove  the  cream.  It  is  well  to  filter  the  milk  through 
a  thin  layer  of  absorbent  cotton  to  remove  any  masses  of 
cream.  The  reaction  should  be  tested  and  if  strongly  acid 
it  should  be  rejected  or  made  1.5%  acid  to  phenolphthaleni 
by  the  addition  of  n/ 1  sodium  hydrate.  Distribute  the  skimmed 
milk  in  small  test  tubes  (7  c.c.  in  each)  and  sterilize  by  dis- 
continuous steaming  in  the  same  manner  and  for  the  same 
length  of  time  as  the  potatoes.  Isabel  and  store  in  locker. 

§  49.  Preparation  of  litmus  milk  for  a  culture  medi- 
um. This  is  prepared  the  same  as  the  milk  medium  with  the 
addition  of  enough  of  an  aqueous  solution  of  litmus  to  impart 
a  decidedly  blue  color  to  the  milk.  Sterilize  label  and  store 
the  same  as  the  milk.  The  litmus  solution  will  be  furnished. 

§  50.  Preparation  of  glucose  agar.  Prepare  100  c.c.  of 
agar  (§  16).  Reserve  one  half  of  it  for  glycerine  agar  and 
to  the  other  half  add  i  °/o  glucose.  Dissolve  the  powdered 
glucose  in  about  5  c.c.  of  boiled,  hot  water  before  adding  it 
to  the  liquid  agar.  After  thoroughly  mixing  distribute  it 
in  small  sterile  test  tubes.  Sterilize,  label,  and  store  the 
same  as  ordinary  agar. 

§  51.  Preparation  of  glycerine  agar.  Take  the  balance 
of  the  agar  prepared  above  (§  50)  and  add  5^  of  pure 
glycerin.  Thoroughly  mix  it  with  the  liquid  agar,  after  which 
distribute  it  in  tubes.  Sterilize,  label,  and  store  as  ordi- 
nary agar. 


37 

§52.  Preparation  of  glucose  bouillon.  This  is  used  in 
the  fermentation  tube.  Take  xooc.c.  of  peptonized  bouillon 
(§  n)  and  add  i  gram  of  pure  grape  sugar  (glucose). 
After  it  is  dissolved  and  thoroughly  disseminated  through 
the  bouillon  by  stirring  or  pouring,  distribute  the  bouillon 
in  the  fermentation  tubes,  filling  completely  the  closed 
branch  and  the  open  bulb  about  half  full.  Sterilize  it  by 
discontinuous  steaming  for  20  minutes  each  day  for  three 
consecutive  days.  It  should  be  labeled  and  kept  in  the 
locker  until  needed  for  use. 

§  53.  Preparation  of  lactose  bouillon.  This  is  prepared 
by  adding  i  °/o  lactose  to  the  peptonized  bouillon.  It  is 
necessary,  however,  that  the  bouillon  used  does  not  contain 
muscle  sugar.  Bouillon  free  from  muscle  sugar  can  usually 
be  obtained  by  macerating  the  meat  for  from  12  to  18  hours 
(§9)  at  a  low  temperature.  After  adding  the  lactose  and 
thoroughly  mixing  it  in  the  bouillon,  sterilize,  label,  and 
store  the  same  as  the  glucose  bouillon. 

§  54.  Saccharose  bouillon.  This  is  peptonized  bouillon 
to  which  i  u/o  saccharose  has  been  added.  It  is  prepared 
from  bouillon  free  from  muscle  sugar,  in  the  same  manner 
as  lactose  bouillon. 


EXERCISE  XL 

INOCULATING  SPECIAL  MEDIA  AND  EXAMINING  CULTURES. 

§55.  General  Directions.  Inoculate  a  tube  of  potato,  one 
of  milk,  one  of  litmus  milk,  one  of  glucose  agar,  a  fermenta- 
tion tube  of  glucose,  one  of  lactose  and  one  of  saccharose 
bouillon.  Label  each  and  place  all  of  them  in  the  incubator. 

Examine  microscopicall)'  the  agar  and  bouillon  cultures 
made  from  the  colony  on  the  agar  plate  (§  41).  Examine 
and  carefully  describe  the  bacteria  from  each  culture  in,  (i) 


38 

a  hanging-drop  preparation,  and  (2)  stained  cover-glass 
preparations.  Stain  a  preparation  with  alkaline  methylene- 
blue,  one  with  carbol  fuchsin,  and  one  with  an  aqueous 
solution  of  methyl-violet.  Make  a  careful  comparison  of 
the  three  preparations  and  note  any  difference  in  the  appear- 
ance of  the  bacteria  or  in  the  degree  of  intensity  of  the 
stain.  Preserve  as  a  permanent  specimen,  to  accompany  the 
notes,  a  preparation  stained  with  each  of  the  dyes. 

Prepare  the  aqueous  solution  of  methyl-violet  (§33). 

§  56.  The  inoculation  of  glucose  agar  to  determine 
the  power  of  the  organism  to  produce  gas.  Boil  the  tube 
of  glucose  agar  in  an  open  water  bath  until  it  is  liquefied,  then 
cool  it  down  to  a  temperature  of  45°  C.  and  inoculate  it  with 
a  loopful  of  the  culture,  carefully  stir  the  agar  with  the  loop, 
after  which  solidify  the  agar  as  quickly  as  possible.  I/abel 
and  stand  in  the  incubator. 


EXERCISE   XII. 
THE  EXAMINATION  OF  CULTURES  ON  SPECIAL  MEDIA. 

§  57.  Explanatory  note.  As  certain  of  these  media  are 
used  to  determine  the  effect  of  the  bacteria  upon  them  it  is 
important  to  observe  very  carefully  not  only  the  appearance 
of  the  growth  of  the  bacteria,  but  also  their  effect,  if  any, 
upon  the  medium  upon  or  in  which  they  are  growing.  This 
is  especially  noticeable  in  the  milk,  litmus  milk  and  sugar 
bouillon  cultures.  The  changes  here  are  largely  due  to  the 
action  of  the  bacteria  on  the  sugars  or  their  power  to  pro- 
duce alkali. 

§  58.  General  Directions.  Examine  and  describe  the 
cultures  made  on  the  special  media  in  Exercise  XI. 

Examine  the  bacteria  on  the  potato  culture  microscopically 
(i)  in  the  fresh  condition  (hanging-drop  preparation),  and 


39 

(2)  in  stained  cover-glass  preparations.  Stain  a  preparation 
with  carbol  fuchsin  and  one  with  an  aqueous  solution  of 
gentian-violet.  Describe  the  appearance  of  the  bacteria 
(§  33)  and  make  a  drawing  of  a  few  of  them. 

§  59.  A  few  points  to  be  observed  in  studying  cultures 
on  special  media,  (a)  Potato.  Note  carefully  the  extent 
and  color  of  the  growth  and  also  its  consistence. 

(£)  Milk.  Note  whether  or  not  the  general  appearance 
and  odor  of  the  milk  has  been  changed,  observe  whether 
the  casein  has  been  coagulated  giving  a  firm  solid  coagulum, 
or  precipitated.  Is  the  coagulum  covered  with  a  liquid 
(serum),  if  so,  is  it  clear  or  milky?  Is  there  any  appear- 
ance suggestive  of  saponification.  Determine  its  consist- 
ence, chemical  reaction  as  indicated  by  litmus  paper  (§  24), 
and  give  as  descriptive  a  name  as  possible  to  its  odor. 

(c)  Litmus  milk.     Note  especially  whether  there  has  been 
any  change  in  color  since  inoculation.     Observations  similar 
to  those  on  the  plain  milk  should  also  be  made. 

(d)  Glucose   agar.     Note  the  character  and   number  of 
colonies  within  the  agar,  and  the  presence,  if  any,  of  gas 
bubbles.     Are  there  few  or  many  of  them  ? 

(*)  Glucose  bouillon.  Observe  the  character  of  the  growth 
(whether  the  liquid  is  faintly  or  heavily  clouded,  turbid, 
contains  flakes,  etc.)  in  (i)  the  open  branch  and  (2)  the 
closed  branch  of  the  fermentation  tube.  Note  the  presence 
or  absence  of  a  membrane  on  the  surface  of  the  liquid  in 
the  open  bulb.  Is  there  a  sediment  in  the  bottom  of  the 
tube  ?  If  so,  describe  its  general  appearance  and  consist- 
ence. Note  the  presence  or  absence  of  gas  in  the  closed 
branch.  Indicate  the  quantity.  Test  the  reaction  of  the 
liquid  with  litmus  paper. 

(/)  Lactose  and  saccharose  bouillon.  Same  as  glucose 
bouillon.  These  cultures  should  be  observed  from  day  to 
day  and  note  made  of  any  changes  which  have  occurred. 


4o 

The  fermentation  tubes  are  used  to  enable  one  to  deter- 
mine the  quantity  and  kinds  of  gases  produced  and  also  the 
anaerobic  properties  of  the  organism. 


EXERCISE  XIII. 
THE  EXAMINATION  OF  CULTURES  (continued). 

§  60.  General  Directions.  Re-examine  the  cultures  made 
on  special  media  and  make  notes  on  all  changes  which  have 
occurred  in  their  appearance. 

Examine  microscopically  in  hanging-drop  preparations 
the  bacteria  from  the  glucose-bouillon  culture. 

Make  3  stained  cover-glass  preparations  from  the  milk 
culture.  Stain  with  the  different  dyes  already  used. 

Re-examine  all  of  the  cultures  previously  made  and  make 
careful  notes  of  any  changes  in  appearance. 

Reject  all  of  the  cultures  made  excepting  those  on  agar 
from  the  colony  which  should  be  preserved  and  clean  the 
tubes  and  Petri  dishes  (§  2  f.). 

§  61.  Making  cover-glass  preparations  from  milk  cul- 
tures. Spread  as  thin  a  film  as  possible  of  the  milk  culture 
on  the  cover-glass  and  allow  it  to  dry  in  the  air.  Then  pass 
it  3  times  through  a  flame.  After  flaming  immerse  the 
preparation  in  a  watch  glass,  or  other  receptacle,  containing 
a  few  cubic  centimeters  of  ether  to  dissolve  out  the  fat. 
Then  remove  and  after  the  ether  has  evaporated  stain  as 
usual.  The  amount  of  albumen  in  the  milk  will  usually 
cause  a  heavy  background  which  will  require  decolorizing 
with  alcohol  or  weak  acetic  acid. 


EXERCISE  XIV. 

THE  CLASSIFICATION  OF  BACTERIA. 

§  62.  Explanatory  note.  Bacteria  is  a  general  and  pop- 
ular term  used  to  designate  a  large  group  of  microscopic 
plants,  the  Schizomycetes.  These  organisms  which  are  widely 
distributed  in  nature  have  been  classified  into  a  certain  few 
families  and  genera  most  of  which  have  a  large  number  of 
species.  Many  of  these  species  have  been  described,  but 
there  are  many  which  have  not.  In  classifying  the  bacteria 
the  genera  are  based  on  morphologic  characters  while,  as 
a  rule  the  species  are  determined  by  means  of  their  biochemic, 
physiologic,  or  pathogenic  properties.  Several  systems  of 
classification  have  been  proposed  but  the  one  which  seems  to 
be  the  most  satisfactory  is  by  Migula.  This  classification 
utilizes  the  morphology  to  such  good  advantage  that  its 
adoption  seems  desirable.  It  requires,  however,  some  serious 
changes  in  the  accustomed  nomenclature.  The  restoration 
of  the  genus  Bacterium,  and  the  assigning  to  it  of  all  non- 
motile,  rod-shaped  organisms  changes  the  genus  of  some  of 
our  most  common  pathogenic  bacteria  from  Bacillus  to  Bac- 
terium. The  most  conspicuous  of  these  are  the  Bacilli  of 
tuberculosis,  glanders,  and  diphtheria,  all  of  which  are  placed 
in  Migula' s  classification  in  the  genus  Bacterium.  The  fam- 
ilies and  genera  recognized  by  him  are  appended. 

FAMILIES. 

I.  Cells  globose  in  a  free  state,  not  elongat- 
ing in  any  direction   before   division 

into  i,  2  or  3  planes i.  Coccaceae. 

II.  Cells  cylindrical,  longer  or  shorter,  and 
only  dividing  in  one  plane,  and 
elongating  to  twice  the  normal  length 
before  the  division. 

(i)  Cells  straight,  rod-shaped,  without 
sheath,  non-motile,  or  motile  by  means 
of  flagella 2.  Bacteriaceae. 


42 

(2)  Cells  crooked,  without  sheath 3.  Spirillaceae. 

(3)  Cells  enclosed  in  a  sheath 4.  Chlamydobacteriaceae. 

(4)  Cells  destitute  of  a  sheath,  united  into 
threads,  motile  by  means  of  an  undu- 
lating membrane 5.  Beggiatoaceae. 

GENERA. 
i.  Coccaceae. 

Cells  without  organs  of  motion. 

a.  Division  in  one  plane i.  Streptococcus. 

b.  Division  in  two  planes 2.  Micrococcus. 

c.  Division  in  three  planes 3.  Sarcina. 

Cells  with  organs  of  motion. 

a.  Division  in  two  planes 4.  Planococcus. 

b.  Division  in  three   planes 5.  Planosarcina. 

2.  Bacteriaceae. 

Cells  without  organs  of  motion i.  Bacterium. 

Cells  with  organs  of  motion  (flagella). 

a.  Flagella  distributed  over  the  whole 

body 2.  Bacillus. 

b.  Flagella  polar 3.  Pseudomonas. 

j.  Spirillaceae. 

Cells  rigid,  not  snake-like  or  flexuous. 

a.  Cells  without  organs  of  motion i.  Spirosoma. 

b.  Cells  with  organs  of  motion  ( flagella ) . 

1.  Cells  with  i,  very  rarely  2-3  polar 

flagella 2.  Microspira. 

2.  Cells  with  polar  flagella-tufts 3.  Spirillum. 

Cells  flexuous 4.  Spirochaeta. 

4.  Chlamydobacteriaceae. 

Cell  contents  without  granules  of  sulphur. 

a.  Cell  threads  unbranched. 

I.  Cell  division  always  only  in  one  plane__i.  Streptothrix. 
II.  Cell  division  in  three  planes  previous  to 
the  formation  of  conidia. 

1.  Cells  surrounded  by   a  very  delicate, 

scarcely  visible  sheath  (marine) 2.  Phragmidiothrix. 

2.  Sheath  clearly  visible  (in  fresh  water) .3.  Crenothrix. 

b.  Cell  threads  branched 4.  Cladothrix. 

Cell  contents  containing  sulphur  granules_5.  Thiothrix. 


43 

5-  Beggiatoaceae. 

Only  one  genus  known  (Beggiatoa  Trev. )  which  is  scarcely  separ- 
able from  Oscillaria.  Character  as  given  under  the  family. 

Of  the  genera  the  Streptococcus,  Micrococcus,  Bacterium, 
Bacillus,  Microspira  and  Spirillum  contain  the  most  import- 
ant of  the  pathogenic  bacteria.  The  familiar  genus  Staphy- 
lococcus  of  an  older  classification  is  included  in  the  genus 
Micrococcus  by  Migula.  It  is  important  that  the  distin- 
guishing characters  of  these  genera  are  thoroughly  learned. 

§  63.  General  directions.  Read  the  references*  on  the 
morphology  and  classification  of  bacteria. 

lyearn  from  the  text-books  and  lecture  notes  the  more 
essential  elements  in  the  structure  of  bacteria. 

Inoculate  a  tube  of  bouillon  from  a  culture  which  will  be 
furnished  of  each  of  the  following  bacteria  and  place  the 
inoculated  tubes  in  the  incubator. 

(1)  A  streptococcus (Streptococcus ). 

(2)  A  micrococcus (Micrococcus ). 

(3)  A   staphylococcus ( Staphylococcus ). 

(4)  A  sarcina (Sarcina  lutea ). 

(5)  A  non-motile  rod-shaped  organism. (Bacterium ). 

(6)  A  motile,  rod-shaped  organism (Bacillus ). 

( 7 )  A  spiral ,  rod-shaped  organism (  ) . 


EXERCISE   XV. 

IDENTIFYING  GENERA  AMONG  BACTERIA. 

§  64.  General  directions.  Carefully  describe  each  of  the 
bouillon  cultures  made  in  Exercise  XIV. 

Prepare  and  examine  a  hanging  drop  preparation  from 
each  of  the  cultures,  and  describe  the  appearance  (form)  of 


*Migula,  Die  Naturlichen  Pflanzenfamilien,  Lieferung  129,  Leipzic,  1896. 
Migula,  System  der  Bakterien,  1897.  Fischer,  Jahrbucher  fur  wissenschaft- 
liche  Botanik,  Band  XXVII,  Erstes  Heft. 


44 

the  organisms  in  each.  Indicate  the  morphologic  characters 
by  which  each  genus  can  be  differentiated  from  the  others. 

Make  a  cover-glass  preparation  from  each  culture  and 
stain  with  an  aqueous  solution  of  Methyl-violet.  Make  a 
careful  microscopic  examination  of  each  preparation  and 
describe  the  bacteria  in  each. 

Make  careful  notes  on  the  appearance  of  the  bacteria  in 
each  preparation  and  preserve  one  specimen  of  each  to  ac- 
company notes. 

Measure  carefully  with  the  filar  micrometer  the  length 
and  thickness  of  three  individual  bacteria  in  the  stained 
preparation  of  the  bacillus.  Record  the  measurements  in 
microns.  (For  the  use  of  the  micrometer  see  chapter 
on  magnification  and  micrometry  in  "The  Microscope" 
by  Professor  S.  H.  Gage.) 

Inoculate  a  tube  of  agar  and  one  of  gelatin  from  each  of 
the  bouillon  cultures. 


EXERCISE  XVI. 

STUDYING  THE  MORPHOLOGY  OF  BACTERIA. 

§65.  General  directions.  Examine  and  describe  the 
cultures  made  in  Exercise  XV. 

Examine  in  the  fresh  condition  and  in  stained  cover-glass 
preparations  the  bacteria  from  the  agar  cultures  and  make 
careful  notes  on  their  forms.  Stain  with  alkaline  methylene- 
blue  and  carbol  fuchsin.  Measure  a  few  of  the  individual 
bacteria  in  the  preparations  of  the  micrococcus,  and  strep- 
tococcus. 

Inoculate  a  tube  of  agar,  one  of  gelatin  and  one  of 
bouillon  with  Bacillus  subtilis  from  a  culture  furnished  and 
place  the  inoculated  tubes  in  the  incubator. 


45 
EXERCISE  XVII. 

STUDYING  AND  STAINING  SPORES. 

§  66.  Explanatory  note.  In  certain  species  of  bacteria 
and  under  suitable  conditions,  there  appears  within  the 
bacteria  highly  refractory  granules  or  bodies  known  as 
spores.  The  formation  of  spores  is  largely  restricted  to  cer- 
tain species  of  bacilli.  The  spores  are  oval  in  form  and  in 
old  cultures  they  can  often  be  found  outside  of  the  bodies  of 
the  organisms  which  produce  them.  They  possess  the  power 
of  resisting  drying,  heat,  and  unfavorable  environment 
much  longer  than  the  bacilli  themselves.  Ordinarily  they 
do  not  stain  by  the  usual  methods  employed  in  staining 
bacteria  so  that  special  methods  are  required.  Several  pro- 
cesses have  been  proposed,  but  the  one  here  given  seems  to 
be  quite  as  efficient  as  any  of  the  others. 

Bacillus  siibtilis,  or  the  hay  bacillus,  is  one  of  the  most 
widely  distributed  species  of  bacteria.  It  develops  spores 
which  can  be  readily  detected  in  either  the  fresh  or  stained 
preparations  from  cultures. 

§  67.  General  directions.  Examine  and  carefully  de- 
scribe the  three  cultures  of  Bacillus  subtilis. 

Make  a  hanging  drop  preparation  from  the  bouillon  and 
one  from  the  agar  cultures  and  examine  them  microscopi- 
cally. Describe  the  bacilli  and  observe  carefully  the  appear- 
ance of  the  spores  both  within  and  outside  of  the  bacilli. 

Make  a  cover-glass  preparation  from  each  culture  and 
stain  with  alkaline  methylene-blue.  Examine  carefully  and 
note  the  appearance  of  spores  which  remain  unstained. 
Make  a  drawing  of  a  few  of  the  bacteria  containing  spores. 

Make  a  few  (about  3)  cover-glass  preparations  and  stain 
them  for  spores. 

Inoculate  a  tube  of  agar  with  Bacillus  cholerae  suis  and 
place  it  in  the  incubator  for  the  next  exercise. 


46 

§  68.  A  method  for  staining  spores.  Make  a  cover- 
glass  preparation,  dry,  and  flame  as  already  described.  Take 
the  preparation  by  the  edge  with  the  fine  forceps,  cover  the 
film  surface  with  carbol  fuchsin  and  hold  the  preparation 
over  the  gas  flame  until  steam  is  given  off,  then  remove  it 
for  a  few  seconds  and  again  heat  it.  Repeat  the  heating  3 
or  4  times.  After  the  stain  has  acted  for  from  3  to  5  min- 
utes rinse  the  preparation  in  water  and  decolorize  it  by  im- 
mersing it  in  a  watch-glass  containing  about  3  c.c.  of  a  i  °/c 
solution  of  sulphuric  acid  or  95%  alcohol.  After  about  one- 
half  minute  remove  the  preparation  and  rinse  it  thoroughly  in 
water.  If  it  is  not  decolorized  repeat  the  bleaching  process. 
This  removes  the  coloring  matter  from  the  bodies  of  the 
bacteria,  but  leaves  it  in  the  spores.  After  thoroughly 
washing  the  preparation,  counter  stain  it  with  a  saturated 
aqueous  solution  of  methylene-blue  for  about  30  seconds, 
rinse  in  water  and  examine.  The  spores  should  be  stained 
red  (with  the  fuchsin)  and  the  rest  of  the  organism  should 
be  colored  blue. 

There  is  a  very  satisfactory  method  recommended  by 
Moller.  For  this  and  other  methods  for  staining  spores, 
see  text-books  on  bacteriology. 


EXERCISE  XVIII. 

STAINING  THE  FLAGELLA  ON  MOTILE  BACTERIA. 

§  69.  Explanatory  note.  The  motile  bacteria  are  pro- 
vided with  a  variable  number  of  long  hair-like  appendages 
or  flagella.  These  are  invisible  in  the  fresh  preparation  and 
they  do  not  stain  by  the  ordinary  methods.  By  special 
staining  processes,  however,  their  presence  can  be  detected. 
Several  methods  have  been  proposed  for  staining  these  fila- 
ments but  nearly  all  of  them  are  based  on  the  use  of  a 


47 

mordant.  Curiously  enough  the  value  of  each  of  these 
methods  seems  to  rest  largely  in  the  individual  using  them, 
as  some  workers  succeed  with  one  method  while  others  fail 
with  it  but  obtain  excellent  results  with  one  of  the  other 
processes.  Although  the  flagella  are  thought  to  be  the 
organs  of  locomotion  they  do  not  seem  to  be  of  any  special 
morphological  value  in  differentiating  closely  related  species. 
They  are,  however,  elements  in  the  structure  of  motile  bac- 
teria and  their  demonstration  is  much  to  be  desired. 

§  70.  General  directions.  Make  a  cover-glass  prepara- 
tion from  the  growth  on  the  agar  culture  of  Bacillus  cholerae 
suis  made  in  Exercise  XVII  and  stain  it  with  carbol  fuchsin. 
Preserve  this  to  compare  with  preparations  stained  for  the 
purpose  of  demonstrating  the  flagella. 

Clean  about  20  cover-glasses  after  the  special  method  for 
flagella  staining  (§4).  Make  about  10  cover-glass  prepar- 
ations on  these  from  the  agar  culture  and  stain  for  flagella. 
Use  LoefHer's  method  but  if  it  does  not  succeed  the  second 
process  may  be  tried. 

§  71.  Making  cover-glass  preparations  for  flagella 
stain.  Place  2  loopfuls  of  sterilized,  distilled  water  on  the 
center  of  the  cover-glass.  Gently  touch  the  surface  growth 
on  the  agar  culture  with  the  end  of  the  platinum  needle  and 
immerse  it  in  the  water  on  the  cover-glass  without  spreading 
the  drop.  The  impregnated  needle  should  carry  bacteria 
enough  for  3  or  4  preparations.  Then  place  the  tray  of 
cover-glasses  in  the  incubator  to  dry.  The  bacteria  become 
disseminated  throughout  the  water  by  means  of  their  power 
of  locomotion.  When  dry  they  are  ready  for  the  staining 
treatment. 

§  72.  Staining  the  flagella  by  Loeffler's  method.  The 
bacteria  are  fixed  to  the  cover-glasses  by  holding  them,  film 
upward,  between  the  thumb  and  fore  finger,  over  a  gas  flame 


48 

for  about  a  minute.     They  are  then  treated  with  the  follow- 
ing mordant : 

Tannic  acid,  20%  solution 10  c.c. 

Sulphate  of  iron,  saturated  solution 5  c.c. 

Fuchsin,  saturated  alcoholic  solution I  c.c. 

This  should  be  filtered  before  using. 

Place  the  fixed  cover-glass  preparation  in  a  large  test  tube, 
cover  it  with  the  mordant  and  carefully  heat  over  a  gas  flame 
or  in  a  water  bath  until  steam  is  given  off.  Allow  the  mor- 
dant to  act  for  from  3  to  5  minutes.  Then  remove  the  cover- 
glass  with  a  bent  wire  loop  and  fine  forceps  and  thoroughly 
rinse  it  in  water.  Then  place  it  in  a  similar  tube  and  cover 
it  with  carbol-fuchsin  for  staining.  Heat  this  as  the  mor- 
dant, and  allow  it  to  act  for  from  5  to  10  minutes.  Remove 
the  cover-glass  as  before  and  thoroughly  rinse  in  water.  If 
the  stain  is  too  deep  decolorize  by  rinsing  the  preparation  for 
a  few  seconds  in  alcohol  and  again  in  water.  It  is  then 
ready  for  the  microscopic  examination  either  in  water  or  it 
may  be  allowed  to  dry  and  be  mounted  in  balsam.  If  the 
first  preparation  fails  add  2  drops  of  a  10%  solution  of  sul- 
phuric acid  to  the  mordant. 

The  flagella  should  appear  as  fine,  hair-like  appendages 
radiating  from  the  bacteria. 

§  73.  Staining  the  flagella  by  Van  Ermengem's 
method.  The  films  are  prepared  as  described  above. 
Three  solutions  are  necessary  : 

Solution  A.     (Fixing  bath). 

Osmic  acid,  2%  solution i  part. 

Tannin  10-25%  solution 2  parts. 

Place  the  films  in  this  for  one  hour  at  room  temperature, 
or  heat  over  a  flame  till  steam  rises,  and  keep  in  the  hot 
stain  for  five  minutes.  Wash  with  distilled  water,  then  with 
absolute  alcohol  for  3  to  4  minutes,  and  again  in  distilled 
water,  and  treat  with  Solution  B. 


49 

Solution  B.  (sensitizing  bath)  This  is  a  5  per  cent,  solu- 
tion of  silver  nitrate  in  distilled  water.  Allow  the  films  to 
be  in  this  for  from  2  to  3  minutes.  Then  without  washing 
transfer  to  Solution  C. 

Solution  C.  (reducing  and  strengthening  bath). 

Gallic  acid 5  grams. 

Tannin 3  grams. 

Fused  potassium  acetate 10  grams. 

Distilled  water 350  c.c. 

Keep  in  this  for  i  to  i^  minutes.  Wash,  dry  and  mount. 
It  will  also  be  found  an  advantage  to  use  a  fresh  supply  of 
C  for  each  preparation,  a  small  quantity  being  sufficient. 


EXERCISE  XIX. 

STAINING  TUBERCLE  BACTERIA  ( BACILLI ). 

§  74.  Explanatory  note.  The  tubercle  bacterium  pos- 
sesses the  power  of  retaining  when  stained  the  coloring  matter 
when  treated  with  a  strong  decolorizer  such  as  a  solution  of 
sulphuric  or  nitric  acid.  On  this  account  its  stain  has  a 
high  differential  value  which  is  made  use  of  in  identifying 
this  organism.  Thus,  in  the  examination  of  sputum  in 
cases  of  suspected  tuberculosis,  the  object  is  to  determine  the 
presence  of  tubercle  bacteria.  As  this  organism  is  not  easily 
cultivated  the  staining  process  is  very  largely  depended  upon 
in  making  a  diagnosis. 

§  75.  General  Directions.  Make  three  cover-glass 
preparations  from  a  culture  of  tubercle  (furnished).  Stain 
these  and  carefully  describe  the  appearance  of  the  bacteria 
and  illustrate  them  with  a  few  drawings. 

Stain  a  cover-glass  preparation  of  tubercular  sputum 
(furnished). 

Read  the  directions  in  the  text-books  for  staining  tubercle 
bacteria  (bacilli). 


50 

§  76.  Staining  tubercle  bacteria.  Prepare  the  cover- 
glass  preparations  from  the  culture  or  the  tuberculous  mate- 
rial and  flame  them  as  already  described.  Stain  in  fresh  car- 
bol  fuchsin.  Place  a  few  drops  of  the  stain  on  the  film-side 
of  the  cover-glass  and  hold  it  over  a  flame  with  forceps  until 
steam  is  given  off.  Allow  the  hot  stain  to  act  for  from  3  to 
5  minutes.  Or,  the  preparation  may  be  floated  on  the  car- 
bol  fuchsin  in  a  watch  glass  without  heat.  In  this  case  it  is 
allowed  to  act  for  from  10  to  15  minutes.  The  preparation 
is  then  rinsed  in  water,  and  decolorized  by  treating  it  with 
a  10%  solution  of  nitric  or  sulphuric  acid  for  ^.  to  one 
minute.  It  is  again  rinsed  in  water  when  it  is  ready  for 
examination.  It  can  be  dried  and  mounted  permanently  in 
balsam.  The  tubercle  bacteria  should  be  stained  a  deep 
reddish  color.  All  other  bacteria  or  animal  tissue  in  the 
preparation  should  be  unstained.  If  desired,  a  counter  stain, 
such  as  alkaline  methylene-blue  can  be  used  after  decoloriz- 
ing. That  is,  the  preparation  can  be  again  stained  for  about 
one  minute  in  alkaline  methylene-blue,  rinsed  in  water  and 
examined  as  before  In  these  preparations  the  tubercle  bac- 
teria are  red  and  the  other  organisms  and  cells  are  blue.  A 
counter  stain  is  of  no  value  in  preparations  made  from  pure 
cultures  or  for  simple  diagnostic  purposes.  When  a  counter 
stain  is  desired  Gabbett's  decolorizing  and  counter  staining 
solution  is  very  convenient. 

Formula  : — 

Methylene-blue  (powder)   2  grams. 

10  %  Sulphuric  acid 100  c.  c. 

(Gabbett  recommended  25  %  solution  of  sulphuric  acid.) 
After  staining  with  the  carbol  fuchsin  treat  the  prepara- 
tions with  this  mixture  until  the  film  has  a  faintly  bluish 
tint.     This  solution   decolorizes  and  counter   stains   at  the 
same  time. 


EXERCISE  XX. 

MAKING  CULTURES  OF  ANAEROBIC   BACTERIA. 

§  77.  Explanatory  note.  Anaerobic  bacteria  will  not 
grow  in  the  presence  of  oxygen  (atmosphere)  and  conse- 
quently they  must  be  cultivated  in  a  medium  from  which 
the  air  has  been  expelled,  or  in  the  presence  of  some  neutral 
gas  such  as  hydrogen.  The  known  important  pathogenic 
anaerobic  bacteria  are  those  of  symptomatic  anthrax, 
tetanus  and  malignant  oedema.  These  are  known  as  obli- 
gatory anaerobes  because  they  require  the  absence  of  oxygen. 
Others  such  as  Bacillus  typhosus  are  known  as  facultative 
anaerobes  as  they  will  multiply  in  media  with  or  without 
atmospheric  oxygen. 

There  are  several  methods  of  cultivating  anaerobic  bac- 
teria but  as  a  rule  they  are  difficult  and  can  not  be  easily 
handled  in  a  general  course.  Two  of  the  simpler  processes, 
however,  will  be  tried. 

§  78.  General  Directions.  Inoculate  a  tube  of  agar 
after  L,iborius'  method  for  anaerobic  bacteria  from  the 
culture  furnished. 

Inoculate  2  fermentation  tubes  from  the  same  culture. 
One  of  the  fermentation  tubes  should  contain  sugar  free 
bouillon  (furnished)  the  other  bouillon  containing  i%  glu- 
cose. 

Inoculate  for  study  at  the  next  Exercise  2  tubes  of  liquid 
agar,  (one  plain  and  one  containing  glucose),  a  fermentation 
tube  of  sugar  free  bouillon  and  one  containing  i  %  glucose 
bouillon  with  B.  coli  communis  from  a  culture  furnished. 

§  79.  Liborius'  method.  liquefy  two  tubes  of  agar  and 
carefully  pour  them  together.  After  this  boil  the  medium 
for  at  least  five  minutes  to  expel  the  air,  cool  it  down  to  a 
temperature  of  45°  C.  and  then  inoculate  it  from  the  cul- 


52 

tures  of  anaerobic  bacillus  furnished.  (B. )  after 

which  cool  the  medium  rapidly  until  it  is  set.  In  inocula- 
ting the  tube  insert  the  loop  nearly  to  the  bottom  and  stir 
very  gently.  In  making  th.e  inoculations  care  must  be  taken 
not  to  introduce  air  by  shaking  the  liquid  medium.  Place 
the  culture  in  the  incubator. 

§  80.  The  fermentation  tubes  for  anaerobic  bacteria. 
If  these  tubes  of  bouillon  have  been  properly  sterilized  the 
closed  branch  is  practically  free  from  atmosphere.  The  ob- 
ligatory anaerobe  will  grow  in  the  closed  branch  only,  while 
the  facultative  anaerobe  will  grow  in  both  the  open  and  closed 
parts.  If  the  organism  is  a  gas  producer,  the  gas  will  force 
the  cloudy  liquid  from  the  closed  bulb  into  the  open  one 
clouding  the  otherwise  clear  liquid.  To  avoid  the  possi- 
bility of  error  in  interpreting  these  growths  it  is  well  to  in- 
oculate a  tube  containing  sugar  free  bouillon  in  'which  case 
the  liquid  in  the  open  bulb  should  remain  clear  as  gas  will 
not  be  formed. 

These  tubes  are  of  equal  value  in  testing  obligatory  and 
facultative  anaerobic  organisms. 


EXERCISE  XXL 

EXAMINATION  OF  ANAEROBIC  CULTURES. 

§81.  General  directions.  Examine  and  carefully  de- 
scribe the  appearance  of  the  anaerobic  cultures  made  in  Ex- 
ercise XX. 

With  the  wire  loop  remove  one  of  the  colonies  from  the 
depth  of  the  agar  culture  and  examine  it  microscopically  in 
(a)  a  hanging-drop  preparation,  and  (b)-  a  stained  cover- 
glass  preparation.  Stain  with  carbol  fuchsin.  Examine 
microscopically  in  similar  preparations  the  bacteria  from  one 
of  the  fermentation  tubes.  Describe  the  appearance  of  the 


53 

bacteria  in  each  preparation  and  make  a  drawing  of  a  few 
of  them. 

Note  the  appearance  of  the  cultures  inoculated  for  the 
study  of  the  gas  production. 

Read  carefully  in  the  text-books  the  methods  for  culti- 
vating anaerobic  bacteria. 


EXERCISE  XXII. 

STUDY  OF  THE  GAS  PRODUCTION  BY  BACTERIA. 

§  82.  Explanatory  note.  The  knowledge  of  the  powers 
of  a  given  species  of  bacteria  to  produce  gas  when  grown  in 
a  medium  containing  sugar  is  quite  important.  It  is  desir- 
able to  determine  both  the  quantity  of  gas  and  its  relative 
composition.  Chemical  analyses  have  shown  that,  in  all 
cases  tested,  the  gas  consists  of  a  mixture  of  hydrogen  (H) 
and  carbonic  acid  gas  (CO2)  with  mere  traces  of  other  gases. 
It  is  importart  to  know  also  the  quantity  of  gas  produced 
with  the  various  sugars  especially  glucose,  lactose  and  sac- 
charose. To  determine  simply  whether  an  organism  will 
produce  gas  it  is  only  necessary  to  inoculate  a  tube  of  liquid 
agar  containing  the  sugars  with  it,  but  if  the  quantity  of  gas 
is  to  be  determined  the  fermentation  tube  is  the  most  con- 
venient apparatus  to  use.  In  some  cases  the  gas  formation 
is  one  of  the  most  striking  differential  properties  as  will  be 
seen  in  the  study  of  hog-cholera  and  typhoid  bacilli.  (For 
a  discussion  of  the  gas  production  and  use  of  the  fermenta- 
tion tube  see  article  by  Dr.  Theobald  Smith,  Wilder  Quarter- 
century  book  p.  187). 

§  83.  General  directions.  Examine  and  describe  the 
cultures  in  the  glucose  and  plain  agar  inoculated  in  Exercise 
XX.  Note  the  approximate  size  and  number  of  gas  bubbles 
in  the  glucose  agar,  and  explain  the  cause  of  difference  in 
the  number  of  bubbles  in  the  two  agar  cultures. 


54 

Examine  the  fermentation  tubes  and  indicate  the  quantity 
of  gas,  and  the  ratio  of  gas  to  liquid  in  the  closed  branch. 

Determine  the  ratio  of  CO2  to  H  in  the  gas. 

In  studying  these  cultures  they  should  be  examined  each 
day  and  the  quantity  of  gas  indicated.  Note  the  bubbles  of 
gas  rising  through  the  liquid  to  the  top.  When  the  gas  pro- 
duction has  ceased  the  liquid  begins  to  clear  near  the  surface 
in  the  closed  branch.  The  final  record  should  not  be  made 
until  this  occurs.  The  reaction  of  the  culture  should  be  de- 
termined and  noted. 

Inoculate  a  tube  of  bouillon  and  make  a  series  of  three 
agar  plates  for  Kxercise  XXIII  from  a  mixed  or  impure 
culture,  which  will  be  furnished. 

§  84.  Determine  the  quantity  of  gas.  It  is  desirable 
to  determine  the  quantity  of  gas  collected  in  the  closed  branch 
in  terms  of  the  capacity  of  the  tube.  To  do  this  measure  the 
length  of  the  closed  branch  and  the  length  of  that  portion  of 
the  tube  filled  with  gas.  Thus  if  the  length  of  the  tube  is 
10  cm.  and  the  length  of  the  portion  filled  with  gas  is  3  cm. 
the  gas  fills  -f$  of  the  branch.  This  can  not  be  determined 
until  the  gas  formation  has  ceased  which  sometimes  requires 
several  (4  to  6)  days. 

§  85.  Determine  the  ratio  of  the  CO2  to  H  in  the  gas 
produced.  This  can  be  approximately  determined  by  the 
use  of  caustic  soda.  Remove  the  plug  from  the  fermenta- 
tion tube  and  fill  the  open  bulb  with  a  2.  %  solution  of  caustic 
soda.  Place  the  thumb  tightly  over  the  open  end  of  the 
tube  and  tip  it  up  so  that  the  gas  will  pass  through  the 
liquid  and  come  into  the  open  bulb.  It  is  then  returned. 
This  should  be  repeated  several  times.  Remove  the  thumb 
when  the  open  bulb  is  full  and  the  liquid  will  rush  up  into 
the  closed  branch  to  fill  the  space  occupied  by  the  CO2  which 
has  been  absorbed  by  the  caustic  soda.  Measure  the  portion 
of  the  tube  first  occupied  with  gas  and  now  filled  with  the 
liquid.  This  will  indicate  the  quantity  of  CO2.  The  bal- 


55 

ance  of  the  gas  is  H.  (There  are  also  traces  of  other  gases) . 
Its  explosive  property  can  be  tested  by  filling  the  open  bulb 
with  water,  cover  it  with  the  thumb  and  again  bring  the  gas 
to  the  open  bulb,  hold  it  close  to  a  flame  and  remove  the 
thumb.  A  distinct  explosion  will  be  heard. 

The  ratio  of  CO2  to  H  can  be  determined  from  the  meas- 
urements. Thus  the  total  amount  of  gas  =  5  cm.  The 
amount  absorbed  (CO2)  =  2  cm.  The  remaining  gas  or 
3  cm.  =  the  H.  The  ratio  of  CO2  to  H  is,  therefore,  as  2:3 
or  CO2:H:  12:3. 


EXERCISE  XXIII. 

IDENTIFYING  GENERA  OF  BACTERIA  AND  OBTAINING 
PURE  CULTURES  FROM  COLONIES. 

§  86.  General  directions.  Examine  the  bouillon  cult- 
ures made  from  the  impure  or  mixed  culture  in  both  the 
fresh  condition  and  in  stained  cover-glass  preparations. 
Stain  with  carbol  fuchsin  and  an  aqueous  solution  of  methyl- 
violet.  Describe  the  appearance  of  the  bacteria  in  these 
preparations  and  note  the  number  of  genera  present.  Give 
the  names  of  the  genera  and  note  the  one  which  predomin- 
ates in  numbers. 

Examine  and  carefully  describe  the  colonies  on  the  plate 
cultures.  Determine  the  number  of  different  colonies,  and 
carefully  describe  each. 

Inoculate  a  tube  of  bouillon  from  a  colony  from  each  genus. 
Label  the  inoculated  tube  with  the  name  of  the  genus.  Make 
stained  cover-glass  preparations  from  a  colony  of  each  genus 
Mount  and  label  one  of  these  preparations  to  accompany  the 
notes. 


EXERCISE  XXIV. 

CLEANING  USED   CULTURE  TUBES,    FLASKS,    AND   PETRI 

DISHES. 

§  87.  General  directions.  Describe  the  bouillon  cul- 
tures made  from  the  colonies  on  the  agar  plates  Exercise 
XXIII.  Describe  the  bacteria  in  the  stained  cover-glass 
preparations  and  compare  them  with  the  bacteria  in  the 
preparations  made  from  the  colonies. 

Reject  and  clean  all  tubes  and  Petri  dishes  that  have  been 
used  for  cultures. 

Complete  the  notes  on  all  work  which  has  been  done  dur- 
ing the  fall  term. 

Have  all  apparatus  for  individual  use  inspected  by  the  in- 
structor and  released. 


EXERCISE  XXV. 

PREPARATION  OF  GLASSWARE  FOR  CULTURE  MEDIA. 

§  88.  Explanatory  note.  It  is  desirable  that  the  tech- 
nique in  plugging  and  sterilizing  test  tubes  and  flasks  and  in 
making  culture  media  should  be  thoroughly  understood.  On 
this  account  this  part  of  the  work  of  the  fall  term  is  repeated, 
but  hereafter  in  the  course,  the  janitor  will  clean  all  glass- 
ware and  the  instructor  will  furnish  all  additional  media  re- 
quired. It  will  be  necessary  in  the  following  exercises  to 
overlap  to  a  considerable  extent  by  way  of  inoculating  media 
and  in  making  the  final  examinations  of  cultures. 

§  89.  General  directions.  Plug  and  sterilize  the  test 
tubes  and  flasks  and  sterilize  the  Petri  dishes. 


57 


EXERCISE  XXVI. 

PREPARATION  OF  CULTURE  MEDIA. 

§  90.  General  directions.  Prepare,  after  the  methods 
already  given  (Exercise  III,  IV  and  X)  the  following  media  : 

15  small  test  tubes  of  bouillon. 

10  small  and  15  large  test  tubes  of  agar. 

10  small  and  15  large  test  tubes  of  gelatin. 

5  small  test  tubes  of  glucose  agar. 

5  small  test  tubes  of  milk. 

5  small  test  tubes  of  litmus  milk. 

5  small  or  large  test  tubes  of  potato. 

5  small  test  tubes  of  distilled  water. 

4  Fermentation  tubes  of  glucose  bouillon. 

4  Fermentation  tubes  of  lactose  bouillon. 

4  Fermentation  tubes  of  saccharose  bouillon. 

10  small  test  tubes  of  Dunham's  solution. 

A  tube  of  each  of  these  media  will  constitute  a  ' '  set  of 
media  "  as  subsequently  used  for  convenience  in  these  direc- 
tions. 

§91.  Dunham's  peptone  solution.  This  is  simply  a 
solution  of  peptone  and  sodium  chloride  in  distilled  water. 
The  formula  is  as  follows  : 

Dried  peptone i  gram. 

Sodium    chloride 0.5  gram. 

Distilled  water 100  c.c. 

Dissolve  the  peptone  and  salt  in  the  water  and  distribute  it 
in  the  tubes  (7  c.c.  each)  and  sterilize  the  same  as  bouillon. 
This  is  used  for  making  the  indol  (cholera-red)  test.  A 
sugar  free  bouillon  has  been  found  better  but  for  an  elemen- 
tary course  Dunham's  solution  is  more  desirable.  See  article 
by  T.  Smith,  Journal  of  Experimental  Medicine,  Vol.  II 
(1897),  p.  543. 


EXERCISE  XXVII. 

STUDY  OF  CERTAIN  PYOGENIC  BACTERIA. 

§  92.  Explanatory  note.  There  are  a  number  of  bacte- 
ria which  are  able  to  cause  suppuration  but  ordinarily  the  for- 
mation of  pus  is  due  to  the  presence  of  streptococci  and  mi- 
crococci.  As  it  is  impossible  to  study  more  than  a  very  few 
of  these  species  two  of  the  most  common  and  one  more  rarely 
{Bacillus  pyocyaneus}  encountered  organism  in  suppurating 
wounds  and  abscesses  are  chosen. 

§  93.  General  directions.  Inoculate  a  "set  of  media  " 
from  each  of  the  cultures  of  the  following  bacteria  which 
will  be  furnished.  Streptococcus  pyogenes,  Micrococcus 
pyogenes  aureus^  and  Bacillus  pyocyaneus  (bacillus  of  green 
pus). 

Read  carefully  the  chapter  on  pyogenic  bacteria  in  the 
text-book. 


EXERCISE  XXVIII. 

PYOGENIC  BACTERIA  (continued). 

§94.  General  directions.  Examine  and  carefully 
describe  the  cultures  made  in  Exercise  XXVII.  Note 
especially  the  growth  on  the  agar,  gelatin,  potato,  and  in 
the  fermentation  tubes.  In  describing  the  color  use  color 
charts  which  are  in  the  laboratory. 

Examine  microscopically  in  (i)  hanging  drop,  and  (2) 
stained  (alkaline-methylene-blue)  cover-glass  preparations 
the  bacteria  from  each  of  the  bouillon  and  agar  cultures. 

Measure  a  few  of  the  bacteria  in  the  stained  preparations 
from  the  agar  cultures  and  make  a  drawing  of  them,  magni- 
fied 1000  diameters. 


59 

Inoculate  for  Exercise  XXIX.,  a  set  of  media  with  B.  coli 
communis  from  a  culture  furnished. 

For  suggestions  in  studying  cultures  and  microscopic 
preparations  of  bacteria,  see  Exercise  VI.  and  XII. 

§95.  Making  drawings  of  bacteria  with  a  definite 
magnification.  In  measuring  the  bacteria  (§64)  we  obtain 
the  dimensions  in  microns  or  in  units  of  i/iooo  of  a  milli- 
meter. In  making  a  drawing,  therefore,  showing  them  mag- 
nified 1000  diameters  it  is  simply  necessary  to  represent  each 
micron  by  one  millimeter.  Thus,  if  the  organism  is  2.5  /x  in 
length  and  i  ^  broad,  the  drawing  should  be  2.5  mm.  long 
and  i  mm.  broad.  If  the  drawing  is  to  represent  the  or- 
ganism magnified  500  diameters  then  each  micron  should  be 
represented  by  0.5  mm.  For  this  purpose  a  metric  rule  and 
a  pair  of  dividers  are  necessary. 


EXERCISE  XXIX. 

BACILLUS  COLI  COMMUNIS. 

§96.  Explanatory  note.  Of  the  bacteria  normally  pres- 
ent on  the  mucous  membranes  of  the  animal  body  the  colon 
group  is,  on  account  of  its  close  morphological  relationship 
to  the  bacilli  of  typhoid  fever  and  hog  cholera,  of  more  than 
ordinary  interest.  There  are  varieties  of  this  organism 
which  approximate  very  closely  in  their  biochemic  properties 
as  well  as  in  their  morphology  the  typhoid  and  also  the  hog- 
cholera  bacilli.  It  is  important  that  this  existing  variation 
is  recognized  and  that  the  list  of  properties  which  characterize 
B.  coli  communis  should  be  clearly  determined.  The  differ- 
entiation of  the  colon  and  typhoid  bacilli,  as  they  exist  in 
nature,  is  one  of  the  difficult  problems  in  practical  bacterio- 
logical work.  The  culture  assigned  approaches  very  closely 
to  the  typical  species. 


6o 

Read  as  far  as  possible  the  chapters  on  this  organism  in 
the  text-books.  Also  article  by  T.  Smith,  The  Am.  Jour,  of 
Med.  Sci.,  Sept.,  1896,  and  by  Adelaide  W.  Peckham,  Jour- 
nal of  Kxp.  Med.,  Vol.  II.,  (1897)  p.  549. 

§  97.  General  directions.  Describe  the  appearance  of 
each  of  the  cultures  of  B.  coli  communis  made  in  Exercise 
XXVIII. 

Bxamine  the  bacteria  in  a  hanging  drop  preparation  from 
the  bouillon  and  glucose  bouillon  cultures. 

Make  and  stain  with  carbol  fuchsin  a  cover-glass  prepara- 
tion from  the  agar  culture.  Measure  a  few  of  the  bacilli  and 
record  their  size  in  the  notes. 

Note  especially  the  quantity  of  gas  formed  in  each  of  the 
fermentation  tubes.  These  cultures  should  be  kept  until  the 
next  exercise  when  they  should  be  examined  again.  If  the 
gas  formation  is  then  completed,  determine  the  quantity  of 
gas  and  the  ratio  of  the  CO2  to  the  H  in  the  gas  in  each  tube. 

Make  two  gelatin  plates  from  the  bouillon  culture.  In 
making  these  plates  use  a  tube  of  sterilized  distilled  water 
for  the  first  dilution. 

Test  the  culture  in  Dunham's  solution  for  the  presence  of 
indol. 

§98.  The  indol  test.  Add  i  c.c.  of  a  .01%  solution 
(fresh)  of  potassium  nitrite  and  a  few  drops  of  concentrated 
sulphuric  acid  to  a  culture  in  Dunham's  solution  or  sugar  free 
bouillon.  A  pinkish  color  indicates  the  presence  of  indol. 
It  is  sometimes  necessary  to  wait  several  hours  for  a  reaction. 
In  an  old  (3  to  5  day)  culture  the  reaction  is  usually  stronger 
than  in  a  more  recently  made  one. 


6i 


EXERCISE  XXX. 

BACILLUS  COLI  COMMUNIS  (continued). 

§  99.  General  Directions.  Re-examine  the  cultures  of 
B.  coli  communis  and  note  any  changes  which  have  occurred  in 
their  appearance.  Determine  the  gas  formula  in  the  fer- 
mentation tubes  of  the  different  sugars. 

Place  the  milk  and  litmus  milk  cultures  in  the  incubator 
and  keep  them  there  for  six  weeks,  noting  the  changes 
which  occur  from  week  to  week. 

Examine  and  describe  fully  the  colonies  on  the  gelatin 
plates.  Preserve  the  plates  and  examine  them  at  the  follow- 
ing two  exercises  and  note  any  changes  which  may  have 
occurred. 

Examine  microscopically,  in  a  stained  preparation,  the 
bacteria  from  a  colony  on  the  gelatin  plate.  Preserve  a  prep- 
aration to  accompany  the  notes. 

Isolate  B.  coli  communis  from  the  intestine  of -an  animal. 
The  intestine  will  be  furnished. 

Inoculate,  for  Exercise  XXXI  a  set  of  media  with  B. 
cholerae  suis  and  one  with  Bacillus  typhosus  from  the  cultures 
furnished. 

§  100.  Isolating  B.  coli  communis.  Carefully  open  the 
intestine  by  a  longitudinal  incision.  Scrape  away  the  con- 
tents, if  any,  from  a  small  area  of  the  mucous  membrane. 
Take  a  loopful  of  the  mucous  from  the  surface  of  the 
mucous  membrane  and  inoculate  a  large  tube  of  liquefied 
gelatin  with  it.  After  shaking  the  tube  carefully  inoculate 
a  second  tube  with  two  loopfuls  from  the  first,  and  a  third 
with  three  loopfuls  from  the  second.  Pour  the  gelatin  into 
Petri  dishes  and  label  them.  These  plates  should  be  exam- 
med  daily.  The  colonies  of  B.  coli  communis  can  be  distin- 
guished from  others  which  may  appear  by  their  thin  spread- 


62 

ing  growth,  sharply  defined  but  irregular  borders  and  their 
bluish  appearance,  especially  with  transmitted  light.  Com- 
pare with  colonies  on  gelatin  plates  made  in  Exercise  XXIX. 


EXERCISE  XXXI. 

BACILLUS  CHOLERAE  SUIS  AND  BACILLUS  TYPHOSUS. 

§101.  Explanatory  note.  The  bacilli  of  typhoid  fever 
and  of  hog  cholera  resemble  each  other  very  closely  mor- 
phologically and  in  certain  of  their  cultural  characters  and 
biochemic  properties.  L,ike  B.  coli  communis  each  of  these 
organisms  has  several  varieties.  Already  several  distinct 
varieties  of  the  hog-cholera  bacillus  have  been  described. 
(The  hog-cholera  group  of  Bacteria.  Bulletin  No.  6,  U.  S. 
Bureau  of  Animal  Industry,  p.  9.)  Certain  of  the  varieties 
of  these  species  approach  each  other  very  closely  while 
others  approach  B.  coli  communis  in  their  various  manifesta- 
tions. It  is  important,  therefore,  that  the  morphology  and 
properties  of  each  of  these  species,  should  be  carefully  de- 
termined. The  fact  should  be  kept  clearly  in  mind  that 
while  these  two  species  and  the  colon  bacillus  resemble  each 
other  in  certain  directions  they  are  so  far  as  has  yet  been 
demonstrated,  distinct  species.  The  special  methods  of 
differentiation  must  be  omitted  from  this  elementary  course. 
Read  carefully  the  chapter  on  Bacillus  typhosus  in  the  text- 
book. 

§  102.  General  directions.  Examine  the  plate  cultures 
made  from  the  intestine  for  the  colon  bacillus. 

Determine  the  approximate  number  of  colonies  on  each 
plate  and  note  especially  the  number  of  colonies  of  B.  coli 
communis  and  describe  their  appearance. 

Inoculate  a  tube  of  agar,  one  of  milk,  and  a  fermentation 
tube  of  glucose  bouillon  from  one  of  the  colonies.  Study 


63 

these  cultures  in  the  next  exercise  and  compare  them  with 
the  notes  on  cultures  of  B.  coli  communis  in  these  media. 

Examine  and  carefully  describe  the  cultures  of  B.  chol- 
erae  suzs,  and  B.  typhosus.  Note  especially  the  reaction  of 
the  cultures  in  the  fermentation  tubes.  Examine  the 
bouillon  cultures  microscopically,  (a)  in  hanging-drop,  and 
(b)  in  stained  (methylene-blue)  cover-glass  preparations. 
Describe  the  appearance  of  the  bacteria. 

Make  a  series  of  three  gelatin  plate  cultures  from  the 
bouillon  culture  of  each  organism. 


EXERCISE  XXXII. 

BACILLUS     CHOLERAE     SUIS    AND     BACILLUS    TYPHOSUS 

( continued  J. 

§103.  General  directions.  Re-examine  all  of  the  cultures 
of  B.  cholera  suis  and  B.  typhosus.  Note  especially  the  condi- 
tion of  the  fermentation  tubes.  Keep  the  milk  and  the  litmus 
milk  cultures  in  the  incubator  for  about  5  weeks  and  note 
any  changes  which  may  take  place  from  week  to  week. 

Examine  and  carefully  describe  the  colonies  on  the  gelatin 
plates. 

Try  the  indol  test  (§  98)  with  the  cultures  in  Dunham's 
solution. 

Make  and  stain  with  alkaline  methylene-blue  a  few  (3  or 
4)  cover-glass  preparations  from  the  organs  (liver,  spleen, 
kidney  or  blood)  of  a  rabbit  which  has  died  from  the  effect 
of  the  inoculation  with  hog-cholera  bacilli.  Note  the  number 
(few  or  many)  of  the  bacteria  in  the  preparations  and  pre- 
serve one  of  them  to  accompany  the  notes.  Make  a  draw- 
ing of  a  few  bacilli. 

Examine  and  complete  the  notes  on  the  culture  of  B.  coli 
communis.  Compare  them  with  the  cultures  of  hog-cholera 
"and  typhoid  bacteria. 


64 

§  104.  Making  cover-glass  preparations  from  tissues. 
With  a  pair  of  fine  forceps  take  up  a  bit  of  tissue  from  the 
freshly  cut  liver,  spleen  or  kidney  and  rub  it  gently  over  the 
surface  of  a  clean  cover -glass.  Care  must  be  taken  that  the 
film  of  tissue  left  on  the  cover-glass  is  thin.  Allow  this  to 
dry  in  the  air  after  which  pass  them  three  times  through  the 
flame  to  fix  the  film  to  the  glass.  They  can  be  stained  the 
same  as  the  cover-glass  preparations  from  the  cultures. 
These  are  often  spoken  of  as  smear  preparations. 

In  making  these  preparations  from  blood,  hold  a  cover- 
glass  by  the  edge  with  a  pair  of  dissecting  forceps.  Place  a 
drop  of  blood  with  the  platinum  loop  on  the  cover-glass  near 
the  forceps.  Take  a  thick,  square  cover-glass  by  the  edge, 
rest  it  on  the  first  above  the  drop  of  blood,  hold  it  at  an 
angle  of  about  20°  from  it  and  draw  it  down  over  the  first 
thus  spreading  the  blood  in  a  very  thin  even  film  over  the 
surface.  If  the  film  is  thick  the  preparation  should  be  re- 
jected and  another  one  made. 


EXERCISE  XXXIII. 

BACILLUS    CHOLERAE    SUIS    AND     BACILLUS     TYPHOSUS 

(continued). 

§  105.  Re-examine  and  complete  the  notes  on  all  of  the 
cultures  except  the  milk  and  litmus  milk  which  should  be 
kept  for  3  weeks  longer.  Carefully  observe  the  reaction  of 
all  the  liquid  cultures. 

Stain  the  flagella  on  the  bacteria  from  the  agar  cultures 

(§72). 

Compare  the  colonies  on  the  gelatin  plates  with  those  of 
B.  coli  communis. 

Make  a  careful  comparison,  in  tabulated  form,  of  the 
morphology,  including  measurements,  of  the  bacilli  them- 


65 

selves  and  of  the  appearance  of  the  growth  in  the  different 
cultures  of  B.  coli  communis  and  the  bacilli  of  hog  cholera 
and  typhoid  fever. 

The  cultures,  excepting  those  in  milk,  can  be  rejected 
now  or,  if  desired,  they  may  be  kept  for  further  study  and 
comparison. 


EXERCISE  XXXIV. 

WIDAL  SERUM  TEST. 

§  1 06.  Explanatory  note.  This  test  depends  upon  the 
fact  that  when  the  blood  serum  of  a  person  suffering  with 
typhoid  fever  or  who  has  recently  recovered  from  it  is  added 
to  a  bouillon  culture  of  the  bacillus,  the  bacilli  become  less 
motile  and  soon  agglutinate  in  small  clumps.  The  dilutions 
used  vary  from  equal  parts  of  serum  and  culture  to  dilutions 
of  i  to  50,000.  It  is  recommended  that  weaker  dilutions 
shall  be  used,  i.  e.,  from  1:10  to  1:50.  The  test  is  believed 
by  many  to  be  possessed  of  much  diagnostic  value.  It  was 
observed  by  Dr.  C.  F.  Dawson  to  apply  to  hog-cholera 
bacilli.  (New  York  Medical  Journal,  Feb.  20,  1897.) 

§  107.  General  directions.  Take  2  loopfuls  of  a  fresh 
bouillon  culture  of  typhoid  bacilli  (which  will  be  furnished) 
and  place  them  on  a  cover-glass,  add  one  loopful  of  blood 
serum  from  a  typhoid  patient  or  the  blood  of  an  immune  guinea 
pig  and  immediately  make  and  examine  a  hanging-drop  prep- 
aration with  a  loopful  of  the  mixture.  Note  the  effect  on  the 
motility  and  the  aggregation  into  clumps.  Specify  the  time 
elapsing  before  the  agglutination  is  well  marked. 

Make  a  similar  examination  of  a  culture  to  which  i/io 
blood  serum  has  been  added. 

Repeat  the  above  test  with  the  blood  from  animals  affected 
with  or  immunized  against  hog  cholera. 


66 

Examine  a  dried  specimen  of  blood  for  this  reaction. 
Add  a  drop  of  bouillon  to  the  drop  of  dried  blood  on  a  slide 
and  after  it  has  become  well  mixed  add  a  loopful  of  it  to  a 
similar  quantity  of  a  fresh  bouillon  culture  and  examine  it  im- 
mediately in  a  hanging-drop. 

§  108.  Securing  blood  for  the  Widal  test,  (i)  Dried 
preparations.  From  a  prick  in  the  finger  or  lobe  of  the  ear  (if 
a  lower  animal  the  shaved  ear  is  a  good  place)  sufficiently  deep 
to  procure  a  drop  of  blood  which  is  placed  on  a  slide  by  means 
of  a  platinum  loop,  and  allow  it  to  dry.  (2)  Serum.  From  a 
similar  but  deeper  prick  or  by  drawing  a  few  drops  of  blood 
from  a  vein  with  a  hypodermic  syringe  secure  a  few  drops  of 
blood,  place  them  in  the  bottom  of  a  small,  short  sterile  tube 
and  allow  the  serum  to  ooze  out.  This  can  often  be  helped  by 
separating  the  blood  from  the  tube  by  means  of  a  sterile  wire. 
If  retained  for  any  length  of  time  before  making  the  test 
the  serum  must  be  kept  in  a  cool  place. 


EXERCISE  XXXV. 

BACTERIUM  (BACILLUS)  SEPTICAEMIAS  HEMORRHAGICAE 
AND  MICROCOCCUS  LANCEOLATUS. 

§  109.  Explanatory  note.  These  organisms  are  the 
causes  of  swine  plague  or  infectious  pneumonia  in  swine  and 
of  croupous  or  lobar  pneumonia  in  man. 

The  name  Bacillus  septicaemiae  hemorrhagicae  was  given 
by  Hiippe  to  the  bacillus  of  swine  plague  (Smith).  This 
bacterium  is  morphologically  and  in  its  cultural  characters 
not  distinguishable  from  the  bacillus  of  rabbit  septicaemia, 
(Koch),  bacillus  of  fowl  cholera,  (Pasteur),  and  of  Schweine- 
seuche,  (Schutz).  It  is  similar  to  a  species  of  pathogenic 
bacteria  found  more  or  less  frequently  in  the  upper  air  pass- 
ages of  nearly  all  of  the  domesticated  animals.  It  is  very 


67 

similar  also  to  a  pathogenic  bacillus  found  in  broncho  pneu- 
monia in  cattle  and  an  infectious  pneumonia  in  sheep.  See 
Report  on  Swine  Plague  by  T.  Smith,  U.  S.  Bureau  of  Ani- 
mal Industry,  1891. 

Micrococcus  lanceolatus  is  the  specific  organism  of 
lobar  pneumonia  in  man.  It  is  found  in  the  pneumonic  lung 
tissue  and  also  in  the  saliva  of  a  certain  number  of  people. 
For  the  history  and  long  synonomy  of  this  organism  see 
article  by  Prof.  Welch  in  the  Johns  Hopkins  Hospital  Bulle- 
tin, Vol.  Ill,  p.  125.  This  organism  resembles  in  many  of 
its  properties  very  closely  that  of  swine  plague.  In  study- 
ing the  two  species  together  there  will  be  good  opportunity 
of  comparing  them  and  detecting  the  differences  and  simi- 
larities existing  between  them. 

§110.  General  directions.  From  the  cultures  furnished, 
inoculate  a  set  of  media,  and  make  a  hanging  drop  and  a 
cover-glass  preparation  from  each. 

Examine  carefully  the  hanging  drop  preparations  and  de- 
scribe the  appearance  of  the  bacteria. 

Stain  the  cover-glass  preparation  with  an  aqueous  solution 
of  methyl-violet,  and  carefully  examine  and  describe  the  bac- 
teria. Measure  a  few  of  them  with  the  filar  micrometer  and 
make  a  drawing  of  a  few  organisms  magnified  1000  diame- 
ters. 

Read  as  far  as  possible  the  articles  cited  on  these  organ- 
isms. 


EXERCISE  XXXVI. 

BACTERIUM  (BACILLUS)  SEPTICAEMAE  HEMORRHAGICAE 
AND  MICROCOCCUS  LANCEOLATUS  (continued.) 

§  in.  General  directions.  Carefully  examine  and  de- 
scribe the  cultures  made  in  Exercise  XXXV. 

Examine  the  agar  and  bouillon  cultures  microscopically  in 
both  the  living  condition  and  in  cover-glass  preparations 


68 

stained  with  alkaline  methylene-blue,  carbol  fuchsin  and  an 
aqueous  solution  of  methyl-violet. 

Describe  the  appearance  of  the  bacteria  and  make  a  draw- 
ing of  a  few  of  them  from  one  preparation. 

Preserve  a  preparation  of  each  species  to  accompany 
notes. 

If  there  is  any  growth  in  the  gelatin  tube  make  a  series  of 
3  gelatin  plates  from  the  bouillon  culture. 


EXERCISE  XXXVII. 

BACTERIUM  (BACILLUS)  SEPTICAEMIAS  HEMORRHAGICAE 
AND  MICROCOCCUS  LANCEOLATUS  (continued). 

§112.  General  directions.  Re-examine  all  the  cultures 
of  these  bacteria  paying  special  attention  to  the  reactions  of 
the  liquid  cultures. 

Make  the  indol  test  with  the  cultures  in  Dunham's  solu- 
tion. 

Make,  stain,  and  examine  a  cover- glass  preparation  from 
an  organ  or  the  blood  of  a  rabbit  which  has  died  from  the 
inoculation  with  swine  plague  bacteria,  and  also  a  prepara- 
tion made  from  a  rabbit  which  has  died  from  the  inoculation 
with  Micrococcus  lanceolatus .  Stain  the  preparations  with  an 
aqueous  solution  of  fuchsin.  Study  the  bacteria  in  these 
preparations  and  carefully  compare  the  two.  Indicate  in 
the  notes  the  differences,  if  any  are  found. 

Keep  the  cultures  until  the  next  exercise  and  compare 
them  again,  after  which  they  may  be  rejected. 


69 
EXERCISE  XXXVIII. 

BACTERIUM  (BACILLUS)  OF  TUBERCULOSIS. 

§  1 13.  Explanatory  note.  The  tubercle  bacterium  does 
not  grow  readily  on  the  ordinary  media.  For  its  cultivation 
blood  serum,  glycerine  agar,  or  bouillon  containing  5  to  7% 
glycerine  are  ordinarily  used.  It  is  with  much  difficulty 
that  it  is  made  to  grow  from  lesions  in  tuberculous  animals, 
but  when  a  culture  is  once  started  it  can,  on  the  media  men- 
tioned above,  and  sometimes  on  agar,  be  cultivated  in  sub- 
cultures with  comparative  ease.  It  grows  very  slowly  and 
it  is  necessary  that  the  temperature  should  be  kept,  without 
variation,  at  about  37°  C.  On  account  of  these  difficulties 
it  is  not  practicable  in  a  general  course  to  cultivate  this  or- 
ganism, but  cultures  on  solid  and  liquid  media  will  be  fur- 
nished by  the  instructor  for  examination.  It  is  important, 
however,  to  be  able  to  recognize  this  organism  in  tissues  and 
sputum  and  consequently  the  following  additional  exercise 
in  staining  and  study  ing  it  is  given. 

§114.  General  directions.  Examine  and  carefully  de- 
scribe the  appearance  of  the  cultures  of  the  tubercle  bacterium 
on  glycerine  agar,  and  in  glycerine  bouillon,  furnished. 

Make  cover-glass  preparations  from  the  culture  furnished 
for  that  purpose,  and  stain. 

Make  4  cover-glass  preparations  from  tuberculous  sputum 
and  .stain  for  tubercle  bacteria.  It  is  often  desirable  to  coun- 
ter stain  the  specimens  from  sputa.  Stain  2  of  them  by 
Gabbett's  method,  and  2  with  carbol  fuchsin  and  decolorize 
without  counter  staining.  Make  a  few  (2  or  3)  cover-glass 
preparations  from  the  liver  or  spleen  of  a  guinea  pig,  which 
has  died  from  tuberculosis  and  stain  them  for  tubercle  bac- 
teria. Stain  one  with  carbol  fuchsin  and  decolorize  with 
sulphuric  acid,  and  stain  one  by  Gabbett's  method,  and 
counter-stain. 


70 

Measure  the  tubercle  bacteria  in  one  of  the  preparations 
and  make  a  drawing  showing  a  few  of  them  magnified  1000 
diameters. 

§  1 15.  Making  cover-glass  preparations  from  sputum. 
Select  the  little  yellowish  colored  masses,  if  present,  remove 
them  by  means  of  the  fine  forceps  or  platinum  loop,  and 
spread  them  on  the  cover-glass  in  a  thin  layer.  If  the  sputum 
is  homogeneous  make  the  preparations  the  same  as  from  cul- 
tures, using  a  small  loopful  of  the  liquid.  If  the  sputum  is 
viscid  it  is  necessary  to  use  the  forceps  to  spread  the  film  on  the 
cover-glass.  After  drying,  the  films  are  fixed  by  passing 
the  preparations  through  the  flame,  after  which  they  are 
ready  to  be  stained. 

§116.  Gabbett's  method  of  staining  tubercle  bac- 
teria. 

(1)  The  stain  (carbol  fuchsin)  : 

Fuchsin, i  gram. 

Absolute  alchool, 10  c.  c. 

5%  carbolic  acid 100  c.  c. 

(2)  The  decolorizer  and  counter  stain  : 

Methylene-blue  powder 2  grams. 

10%  sulphuric  acid, 100  c.  c. 

Stain  the  preparation  with  the  first  solution  as  described 
in  (§  76)  then  rinse  in  water  and  stain  one  minute  with 
the  second  solution  which  decolorizes  and  counter  stains  at 
the  same  time,  and  again  rinse  in  water.  If  the  film  has  a 
bluish  tint  it  is  ready  for  examination,  if  not,  it  should  be 
stained  a  little  longer  in  the  second  solution.  In  these  prep- 
arations the  tubercle  bacteria  should  appear  as  slender,  more 
or  less  curved,  rod-shaped  bodies  of  a  deep  reddish  color  while 
the  surrounding  tissue  and  other  bacteria  present  are  stained 
a  more  or  less  intense  blue. 


EXERCISE  XXXIX. 

BACTERIUM  (BACILLUS)  MALLEI. 

§  117.  Explanatory  note.  This  organism  grows  most 
characteristically  on  potato,  and  somewhat  feebly  in  the 
other  media  heretofore  used.  It  develops  readily  on  acid 
agar,  and  in  acid  glycerine  agar  and  acid  glycerine  bouillon. 
For  this  reason  it  is  not  inoculated  into  the  full  set  of  media. 
In  diagnosing  glanders,  it  is  customary  to  inoculate  guinea- 
pigs  with  the  suspected  material.  From  the  lesions  in  these 
animals,  if  the  disease  develops,  pure  cultures  can  usually  be 
.obtained.  It  can  be  identified  from  its  morphologic  and 
cultural  characters. 

§  118.  General  directions.  Inoculate  a  tube  of  potato, 
one  of  agar,  one  of  acid  agar,  one  of  acid  glycerine  agar,  one 
of  glucose  agar  and  one  of  bouillon  from  a  culture  furnished. 
(The  special  media  here  introduced  will  be  furnished  by  the 
instructor). 

§  119.  The  preparation  of  acid  agar.  This  is  prepared 
the  same  as  ordinary  agar  (§  16)  with  the  omission  of  the 
sodium  hydrate  in  the  bouillon  from  which  it  is  made. 

§  1 20.  The  preparation  of  acid  glycerine  agar.  Add 
5%  glycerine  to  acid  agar  before  sterilizing  it. 


EXERCISE  XL. 

BACTERIUM  (BACILLUS)  MALLEI,  (continued). 

§121.  General  directions.  Examine  and  carefully  de- 
scribe all  the  cultures  of  Bacterium  mallei. 

Make  three  cover-glass  preparations  from  the  acid  agar 
and  from  the  bouillon  cultures,  and  stain  one  of  each  with 
alkaline  methylene-blue,  one  with  carbol  fuchsin  and  one 


72 

with  an  aqueous  solution  of  methyl-violet.  Describe  the 
bacteria  and  make  a  drawing  of  a  few  of  them.  Preserve 
one  preparation.  Keep  the  cultures  and  re-examine  them 
at  each  of  the  three  following  exercises.  Note  especially 
the  character  and  color  of  the  growth  on  the  potato. 


EXERCISE 
ACTINOMYCOSIS. 

§122.  Explanatory  note.  Although  actinomycosis  is  not 
a  bacterial  disease,  it  is  of  peculiar  interest  as  it  is  caused 
by  a  fungus  (ray  fungus)  of  higher  rank  than  bacteria.  It 
occurs  in  man  and  certain  of  the  lower  animals.  The  ray 
fungus  can  be  cultivated  on  glycerine  agar  and  certain  other 
media  but  ordinarily  it  can  be  easily  detected  in  fresh  or 
stained  preparations  of  the  affected  tissue. 

§  123.  General  directions.  Examine  a  culture  of 
actinomycosis  furnished  and  describe  its  appearance.  Make 
a  hanging  drop  and  a  stained  cover-glass  preparation  from 
it.  Examine  and  make  a  drawing  of  the  fungus. 

Examine  sections  (which  will  be  furnished)  of  animal 
tissues  containing  the  ray-fungus  and  also  permanent  prepa- 
rations of  the  fungus  isolated  by  maceration  from  actinomy- 
cotic  tissue. 

Inoculate  a  set  of  media  with  Bacterium  anthracis  for 
studying  at  the  next  exercise. 


EXERCISE   XLIL 

BACTERIUM    (BACILLUS)   ANTHRACIS. 

§  124.  General  directions.  Examine  and  describe  each 
of  the  cultures  of  this  organism  inoculated  in  the  last 
Exercise. 


73 

Examine  microscopically  the  bouillon  and  agar  cultures 
in  both  hanging  drop  and  stained  cover-glass  preparations. 
Stain  a  preparation  with  each  of  the  three  ordinary  staining 
solutions  heretofore  used. 

Measure  a  few  of  the  bacteria  in  a  stained  preparation  and 
make  a  drawing  of  them  magnified  1000  diameters. 

Make  a  series  of  3  agar  plates  from  the  bouillon  culture. 

Examine  sections  of  animal  tissue  containing  anthrax 
bacteria.  Make  and  examine  a  few  cover-glass  preparations 
from  the  liver  of  an  animal  (guinea-pig  or  rabbit)  which 
has  just  died  of  anthrax.  (This  will  be  furnished  by  the 
instructor). 


EXERCISE   XLIII. 

BACTERIUM    (BACILLUS)    ANTHRACIS    (continued). 

§125.  General  directions.  Re-examine  all  of  the  cul- 
tures of  Bacterium  anthracis  and  describe  any  changes  in 
their  appearance  which  may  have  taken  place. 

Examine  the  agar  culture  for  spores  in  (i)  a  hanging- 
drop  preparation,  and  (2)  a  stained  cover-glass  preparation. 
Describe  the  appearance  of  the  bacteria  and  spores  in  a 
preparation  from  each. 

Stain  the  spores  (Exercise  XVII.) 

Study  and  describe  the  appearance  of  the  colonies  on  the 
agar  plates.  Make  an  outline  drawing  of  a  few  of  the  sur- 
face and  deep  colonies. 

Reject  all  cultures  except  the  agar  plates  which  may  be 
kept  until  the  next  exercise  for  further  observation  before 
rejecting.  (These  cultures  should  be  put  in  charge  of  the 
instructor  who  will  see  that  the  spores  are  destroyed  before 
the  tubes  are  cleaned). 

Inoculate  a  set  of  media  with  Bacterium  diphtheriae ,  for 
study  at  the  next  exercise. 


74 
EXERCISE   XUV. 

BACTERIUM  (BACILLUS)  DIPHTHERIAS. 

§  126.  Explanatory  note.  The  bacterium  of  diphtheria 
is  often  called  the  Klebs-L,oeffler  bacillus.  It  is  the  specific 
cause  of  diphtheria  in  man  but  it  is  not,  so  far  as  known,  the 
cause  of  diphtheria  in  pigeons  and  poultry.  It  is  found  in 
the  throat  of  people  suffering  with  diphtheria,  but  it  is  not 
found  ordinarily  elsewhere  in  the  bod}7.  Its  appearance  in 
the  throat  lesions  is  availed  of  in  diagnosing  the  disease. 
For  this  reason  it  is  especially  important  that  its  morphology 
as  well  as  its  cultural  characters  should  be  carefully  deter- 
mined. Although  this  organism  grows  on  nearly  all  of  the 
media  commonly  used,  its  development  is  more  rapid  and  its 
growth  more  characteristic  on  L,oeffler's  blood  serum.  The 
bacterium  of  diphtheria  seems  to  be  modified  in  its  morphol- 
ogy in  growing  on  different  media  more  than  any  of  the 
other  pathogenic  bacteria.  Particular  attention  should  be 
given  to  its  morphology  and  staining  properties. 

§  127.  General  Directions.  Examine  and  describe  the 
cultures  made  in  Exercise  XLIII. 

Examine  the  agar  and  buillon  cultures  microscopically  in 
(i)  hanging-drop  preparations,  and  (2)  stained  cover-glass 
preparations.  Stain  with  alkaline  methylene-blue  and  with 
carbol  fuchsin.  Also  stain  a  preparation  after  Gram's 
method. 

Inoculate  a  tube  of  glycerine  agar,  one  of  blood  serum,  and 
one  of  Loeffler's  blood  serum  from  the  agar  culture. 

The  blood  serum  and  Loeffler's  serum  necessary  for  this 
exercise  will  be  furnished. 

§  128.  The  preparation  of  blood  serum.  When  a 
small  quantity  is  sufficient  it  can  be  obtained  from  a  dog 
aseptically.  The  animal  is  properly  tied  on  the  operating 
table,  etherized,  the  skin  over  the  carotid  or  femoral  artery 


75 

is  thoroughly  disinfected  and  turned  back,  the  artery  ex- 
posed, a  sterile  glass  canula  inserted  and  the  blood  collected, 
by  means  of  a  sterile  rubber  tube  attached  to  the  canula,  in 
a  sterile  flask.  After  the  serum  is  formed  it  can  be  drawn 
off  with  a  sterile  pipette,  and  distributed  in  small  sterile  test- 
tubes  (5 — 7  c.c.  in  each).  It  is  well  to  set  the  liquid  serum 
in  an  incubator  for  a  few  days  to  test  its  sterility.  The  tubes 
of  liquid  serum  are  inclined  (the  same  as  agar)  and  placed 
in  a  blood  serum  sterilizer,  or  other  chamber  in  which  the 
temperature  can  be  raised  to  70°  or  75°  C.,  and  kept  there 
until  the  serum  has  set.  Label  and  store. 

If  larger  quantities  of  the  blood  are  required,  it  is  more 
convenient  to  collect  it  from  the  bleeding  animals  (cattle  or 
sheep)  in  a  slaughter  house.  It  is  often  necessary  to  sterilize 
the  liquid  serum  after  it  has  been  distributed  in  tubes  (5  c.c. 
in  each),  when  it  has  been  collected  in  this  manner.  This 
can  be  done  by  heating  them  in  a  water  bath  at  62°  C.  for 
2  hours  each  day  for  four  consecutive  days. 

§  129.  The  preparation  of  Loeffler's  blood  serum. 
This  consists  of  neutral  bouillon  (prepared  from  meat)  con- 
taining i  %  glucose,  i  part,  liquid  blood  serum  3  parts. 
Mix  and  distribute  in  sterile  test  tubes,  incline  and  solidify 
the  same  as  blood  serum.  The  temperature  should  be  about 
75°  C.  and  the  exposure  will  be  necessarily  longer  than  for 
the  pure  blood  serum.  Label  and  .store. 

For  other  methods  of  preparing  serum  and  other  special 
media.  See  text-books. 

§  130.  Staining  bacteria  by  Gram's  method.  Prepare 
the  cover-glass  preparations  as  already  described.  Stain 
them  in  gentian-violet  aniline  water  or  in  a  saturated  alco- 
holic solution  of  gentian  violet  in  5  %  carbolic  acid  in  the 
proportion  of  i  to  20  for  from  5  to  7  minutes.  Rinse  in 
water  and  transfer  them  to  a  watch  glass  containing  Gram's 
solution  until  the  color  becomes  quite  black.  This  requires 
from  one  to  two  minutes  ;  then  place  the  preparations  in  a 


76 

watch  glass  containing  alcohol  and  allow  them  to  remain 
there  until  the  color  has  almost  entirely  disappeared,  or  has 
become  a  pale  gray.  Rinse  in  water  and  examine  at  once, 
or  allow  them  to  dry  and  mount  in  balsam.  (Sections  of 
tissues  must  be  dehydrated  and  cleared  before  mounting. ) 
Formula  for  Gram's  solution  (L,u gal's)  : 

Iodine I  gram. 

Potassium  iodide 2  grams. 

Distilled  water 300  c.  c. 

Certain  bacteria  stain  deeply  when  treated  by  this  method 
while  others  are  decolorized  by  the  alcohol.  On  this  account 
it  is  of  some  differential  value. 


EXERCISE  XLV. 

BACTERIUM  (BACILLUS)  DIPHTHERIAE    (continued). 

§  131.  General  Directions.  Re  examine  all  the  cultures 
of  B.  diphtheriae,  and  describe  all  changes  which  have  ap- 
peared in  them. 

Examine  microscopically,  in  stained  cover-glass  prepara- 
tions, the  bacteria  from  the  glycerine  agar,  blood  serum,  and 
Loeffler's  blood  serum  cultures.  Stain  with  alkaline  methy- 
lene-blue,  and  note  especially  the  way  the  bacteria  stain. 
Stain  a  few  preparations  with  the  other  staining  solutions 
and  compare  with  the  methylene-blue  stain. 

Note  with  special  care  the  morphology  of  the  bacteria  and 
make  a  drawing  of  a  few  of  them. 

Examine  sections  of  diphtheritic  membrane  showing  diph- 
theria bacteria,  and  streptococci  (furnished).  Reject  all 
cultures  of  diphtheria  bacteria. 

Inoculate  from  the  unknown  cultures  furnished  such  media 
as  the  requirements  of  the  next  exercise  seem  to  demand. 


•  77 


EXERCISE 

DETERMINE  THE  SPECIES  OF  BACTERIA. 

§  132.  Explanatory  note.  The  two  cultures  of  bacteria 
assigned  belong  to  species  already  studied.  The  student 
should  identify  the  species  of  the  bacteria  in  the  cultures. 
To  do  this  such  media  should  be  inoculated  and  such  micro- 
scopic examinations  made  as  he  thinks  necessary.  The 
notes  should  contain  a  complete  record  of  the  work  and  the 
reasons  for  the  identifications  made. 

§  133.  General  directions.  Identify  the  bacteria  in  the 
cultures  assigned  at  the  last  exercise. 


EXERCISE  XLVII. 

ISOLATING  AND   IDENTIFYING   BACTERIA   FROM   ANIMAL 

TISSUES. 

§  134.  Explanatory  note.  In  making  a  bacteriological 
investigation  into  the  cause  of  death  in  an  animal  or  man  it 
is  necessary  to  make  cultures  from  the  various  organs  and 
the  blood  to  find  whether  or  not  there  are  any  pathogenic  or 
other  bacteria  present.  This  necessitates  a  knowledge  of 
making  cultures  from  animal  tissues.  In  this  exercise  an 
experimental  animal  (rabbit  or  guinea-pig)  will  be  provided 
which  has  died  from  some  bacterial  disease.  The  purpose 
of  this  examination  is  to  find  out  what  that  disease  is.  To 
save  animals  each  student  will  make  cultures  from  but  one 
organ.  Opportunity  will  be  afforded  from  time  to  time 
during  the  course  for  making  cultures  from  various  animal 
tissues. 

§  135.  General  directions.  The  experimental  animal 
will  be  furnished  tied  out  on  a  post-mortem  tray  and  the 


78 

viscera  exposed.  (Directions  for  the  post-mortem  examina- 
tion will  be  given  in  the  course  in  pathology). 

Inoculate  a  tube  of  bouillon,  one  of  agar,  and  a  fermenta- 
tion tube  of  glucose  bouillon,  from  either  the  liver,  spleen, 
or  kidney.  (In  an  actual  investigation  of  an  unknown  dis- 
ease, cultures  should  be  made  from  all  of  the  organs,  blood , 
and  lymphatic  glands). 

Make  a  series  of  3  agar  plate  cultures. 

Make  several  cover-glass  preparations  from  the  organ  from 
which  the  cultures  were  made. 

Stain  and  examine  the  cover-glass  preparations  and  describe 
the  bacteria,  if  any  are  found.  Stain  with  alkaline  methy- 
lene-blue  and  carbol  fuchsin.  (It  is  sometimes  necessary  to- 
preserve  pieces  of  the  tissue  in  alcohol  or  to  fix  them  in 
some  of  the  other  fixing  fluids  for  sectioning  and  staining, 
preparatory  to  studying  them). 

Preserve  one  or  more  of  the  cover-glass  preparations  to 
accompany  the  notes. 

§  136.  Making  cultures  from  animal  tissues.  Heat  a 
platinum  spatula  to  a  red  heat  in  a  gas  flame  and  scorch  the 
surface  of  the  organ.  Flame  a  pair  of  fine  forceps  and  tear 
the  scorched  surface  away,  and  with  the  platinum  loop  take 
up  a  loopful  of  the  tissue  underneath  with  which  inoculate 
the  media.  It  is  also  desirable  to  inoculate  a  tube  of  slant 
agar  with  the  needle  by  drawing  it  over  the  surface  after 
changing  it  with  tissue.  In  making  plate  cultures  use  a 
loopful  of  the  crushed  tissue  for  the  first  tube.  The  quantity 
of  the  tissue  necessary  to  give  a  desired  number  of  colonies 
cannot  be  anticipated,  although  experience  in  working  with 
different  organisms  in  animals  renders  one  able  to- 
approximate  the  amount  required. 


79 


EXERCISE  XLVIII. 

ISOLATING  AND    IDENTIFYING  BACTERIA   FROM  ANIMAL 
TISSUES  (Continued). 

§  137.  General  Directions.  Examine  and  describe  all  of 
the  cultures  made  from  the  animal  tissues. 

Examine  the  bouillon  and  agar  cultures  microscopically  in 
the  fresh  condition  and  in  stained  cover-glass  preparations. 

If  the  species  can  not  be  determined  from  these  cultures 
and  examinations,  make  such  others  from  the  agar  culture  as 
may  be  necessary  to  do  so.  Examine  these  at  the  next  ex- 
ercise when  the  notes  can  be  completed. 

State  in  the  notes  the  facts  upon  which  the  identification 
is  based. 


EXERCISE  XLIX. 

THE  EXAMINATION  OF  SECTIONS  OF  TISSUE  CONTAINING 
BACTERIA. 

§  138.  Explanatory  note.  The  preparation  of  tissues  for 
sectioning  and  the  study  of  the  tissue  changes  more  properly 
belong  in  the  course  in  pathology.  It  is  important,  however, 
that  one  is  able  to  distinguish  bacteria  in  the  lesions  which 
they  produce.  For  this  reason  an  exercise  is  devoted  to  the 
study  of  bacteria  in  sections  of  tissues  already  stained  and 
mounted.  These  include  the  various  pneumonias,  tubercu- 
losis, anthrax,  hog  cholera,  typhoid,  septicaemia,  etc. 

§  139.  General  directions.  Examine  the  sections  fur- 
nished for  the  bacteria  and  note  especially  their  distribution 
in  the  tissues.  Make  drawings  of  a  few  of  the  bacteria  from 
each  preparation. 


8o 


EXERCISE   L. 

BACTERIOLOGICAL  EXAMINATION  OF  PUS  AND  EXUDATES. 

§140.  Explanatory  note.  It  is  often  very  desirable  for 
diagnostic  purposes  to  make  a  bacteriological  examination 
of  the  pus  from  abcesses  and  the  muco-purulent  discharges 
or  exudates  from  mucous  or  serous  membranes. 

Several  diseases  can  be  diagnosed  in  this  way.  It  is  often 
necessary  to  make  cultures  and  it  is  always  advisable  to  do 
so  whenever  the  material  is  in  a  suitable  condition.  Among 
the  specific  diseases  for  which  such  an  examination  is 
especially  valuable  are  actinomycosis,  gonorrhea,  diphtheria, 
and  tuberculosis.  It  is  often  desirable  to  determine  the 
character  of  the  bacteria  in  the  numerous  abcesses  and  sup- 
purating wounds  encountered  in  both  man  and  the  lower 
animals.  These  examinations  will  be  made  from  the  more 
desirable  cases  as  they  appear  from  time  to  time.  In  this 
exercise  such  cover- glass  preparations  will  be  examined  as 
have  been  accumulated  for  this  purpose. 

§  141.  General  directions.  Examine  the  pus  in  the  fresh 
condition  and  note  its  composition,  leucocytes,  red  blood 
corpusles,  fungi  (actinomycosis),  etc. 

Make  cover-glass  preparations  and  stain  one  or  more  of 
them  with  carbol  fuchsin  and  one  with  alkaline  methylene- 
blue  and  examine.  Note  the  celluar  tissue  elements  present 
and  also  describe  the  bacteria  found.  If  the  pus  is  from  a 
case  suspected  to  be  of  a  specific  nature  stain  and  examine 
for  the  corresponding  organism. 

If  actinomycosis,  the  ray  fungus  may  be  seen  better  in  the 
fresh  preparation.  Add  a  drop  of  a  10%  solution  of  caustic 
potash  to  a  loopful  of  pus  on  the  slide  and  cover  with  a 
cover-glass  and  examine. 

If  gonorrheal  discharge,  stain  the  cover-glass  preparations 
with  alkaline  methylene-blue  or  with  carbol  fuchsin.  Note 


8i 

the  appearance  of  the  cocci  both  within  and  outside  of  the 
pus  cells. 

If  from  supposed  tuberculosis,  stain  for  that  organism. 

If  from  diphtheria,  stain  for  that  organism  and  note  the  mor- 
phology of  the  bacteria. 

If  from  the  pus  of  an  abscess,  stain  for  pyogenic  bacteria. 

§  142.  Making  cover-glass  preparations  from  pus. 
Spread  as  thin  a  film  of  the  pus  as  possible  on  the  cover- 
glass.  This  can  be  readily  done  by  drawing  the  edge  of  a 
square  cover-glass  over  the  surface  of  another  cover-glass 
on  which  a  bit  of  the  pus  has  been  placed.  See  method  for 
making  cover-glass  preparations  from  blood  (§  104). 


EXERCISE  IvL 

A    BACTERIOLOGICAL    EXAMINATION   OF    THE   SKIN  FOR 

MICROCOCCUS  (STAPHYLOCOCCUS)  EPIDER- 

MIDIS  ALBUS. 

§  143.  Explanatory  note.  There  is  liable  to  be  on  or  in 
the  skin  a  number  of  bacteria  which  resist  the  ordinary 
methods  of  cleansing,  owing  to  their  being  deep  seated  in 
the  epidermis.  The  most  important  among  these  is  M. 
(Staph.}  epidermidis  albus.  These  organisms  often  infect 
wounds  in  surgical  operations.  An  abrasion  of  the  skin  with 
a  sterile  instrument  may  be  followed  by  the  infection  of  the 
wound  with  this  or  other  species  of  bacteria  which  were  on, 
or  in  the  skin  itself.  The  work  of  this  exercise  is  to  demon- 
strate the  presence  of  these  organisms. 

§  144.  General  directions.  Wash  the  hands  thoroughly 
with  soap  and  water,  using  the  brush.  Then  wash  them  in 
a  solution  of  carbonate  of  sodium  and  rinse  thoroughly  in 
boiled  water  and  wipe  with  a  sterilized  towel. 

With  a  flamed  and  cooled  scapel  scrape  the  epidermis  over 
a  small  area  about  the  finger  nails  and  with  these  scrapings 


82 

inoculate  a  tube  of  bouillon  and  make  a  series  of  2  agar  plate 
cultures. 

At  the  next  exercise  describe  these  cultures  and  examine 
the  colonies  microscopically  to  determine  the  genera  of  bac- 
teria. If  a  micrococcus  which  grows  in  clumps  is  found,  in- 
oculate a  tube  of  agar  with  it  and  at  the  following  exercise 
examine  and  describe  its  appearance.  Indicate  in  the  notes 
the  number  of  colonies  of  bacteria  which  developed  in  the 
plate  cultures  and  the  genera  which  appear  in  the  bouillon 
culture. 


EXERCISE 

DETERMINE  THE  THERMAL  DEATH  POINT  OF  BACTERIA. 

§  145.  Explanatory  note.  It  is  important  to  know  the 
minimum  temperature  which  will  kill  bacteria,  especially  the 
pathogenic  forms.  The  uses  for  such  knowledge  are  numer- 
ous in  practical  disinfection  and  pasteurization.  For  the 
various  methods  employed  in  making  these  determinations, 
see  text  books  and  special  articles  on  this  subject.  The 
method  here  given  and  which  can  be  followed  by  a  full  sec- 
tion of  students  will  give  only  approximate  results.  It 
should  not  vary,  however,  more  than  one  degree  from  the 
actual  thermal  death  point  in  moist  heat. 

§  146.  General  directions.  Inoculates  tubes  of  bouillon 
from  each  of  two  cultures  (It.  subtilis,  old  culture,  and  B. 
typhosus)  furnished. 

In  inoculating  be  sure  not  to  touch  the  sides  of  the  tube 
with  the  inoculating  loop.  The  tubes  should  have  stood  in 
the  water  bath  at  60°  C.  for  at  least  10  minutes  before  they 
are  inoculated. 

Place  one  of  these  tubes  in  the  incubator  for  a  control. 
Stand  the  others  in  a  wire  basket  and  set  them  in  the  thermo- 
regulated  water  bath  which  is  at  60°  C.  The  water  should 


83 

come  just  above  the  liquid  in  the  tubes.  Remove  the  tubes, 
one  of  each  species,  as  follows.  One  in  5  minutes,  one  in  10 
minutes,  one  in  15  minutes  and  one  in  20  minutes.  Isabel 
and  place  them  in  the  incubator. 

At  the  next  exercise,  examine  the  heated  tubes  and  note 
which  are  clear  and  which  contain  a  growth.  If  the  tubes 
heated  for  10  minutes  or  longer  have  a  growth,  repeat  the 
experiment  at  7.0°  C.  If  this  fails  to  destroy  them  repeat  at 
80°  C.  and  if  necessary  apply  a  still  higher  temperature. 

Examine  the  cultures  microscopically  in  all  the  fertile 
tubes  to  determine  if  they  are  pure. 

Explain  the  cause  for  the  difference  in  the  thermal  death 
point  between  the  two  organisms. 

Inoculate  for  the  next  exercise  a  tube  of  bouillon  from 
each  of  the  cultures  of  M.  (Staph.)  pyogenes  aureus  and  B. 
stibtilis  furnished. 


EXERCISE  UII. 

DETERMINING  THE  EFFICIENCY  OF  DISINFECTANTS. 

§  147.  Explanatory  note.  The  efficiency  of  the  more 
commonly  used  disinfectants  has  been  determined  for  most 
of  the  pathogenic  bacteria,  but  new  disinfectants  are  con- 
stantly being  put  upon  the  market,  and  before  it  is  safe  to 
use  or  recommend  them,  their  efficiency  should  be  deter- 
mined. With  many  of  the  disinfectants,  such  as  carbolic 
acid,  corrosive-sublimate,  lime,  and  the  mineral  acids,  much 
stronger  solutions  are  commonly  used  than  are  actually 
necessary  to  kill  the  bacteria,  owing  to  the  fact  that  fre- 
quently it  is  necessary  to  allow  for  an  indefinite  waste  due  to 
the  union  of  the  disinfectant  with  other  substances,  usually 
organic,  with  which  the  bacteria  are  mixed.  For  the  differ- 
ent methods  of  testing  the  efficiency  of  disinfectants,  see 
text-books.  A  very  simple  process  is  given  here. 


84 

§  148.  General  directions.  Prepare  20  c.  c.  of  each  of  a 
.25,  .10,  .02%  solutions  of  formalin  (40%  formaldehyde  in 
water),  and  place  them  in  sterile  tubes,  putting  10  c.  c.  in 
each.  Use  distilled  water  in  making  the  dilutions.  Add  to 
each  of  the  tubes  in  one  set  ^  c.  c.  of  the  bouillon  culture 
of  Micrococcus  {staphy  )  pyogenes.  And  to  each  of  the  tubes 
in  the  other  set,  the  same  quantity  of  the  culture  of  B.  subtilis. 

Use  a  sterile  pipette  for  adding  the  culture  to  the  dis- 
infectant. 

Inoculate  a  tube  of  bouillon  with  six  loopfuls  from  each 
of  these  tubes  after  the  expiration  of  the  following  periods 
of  time,  viz.  :  i  min.,  5  min.,  10  min.,  20  min.,  and  3omin. 
In  making  these  inoculations  allow  the  loop  to  go  to  the  bot- 
tom of  the  inoculated  tube.  Label  each  inoculated  tube  with 
the  strength  of  the  disinfectant  and  time  of  exposure  and 
place  it  in  the  incubator.  It  should  be  noted  that  the  adding 
of  ^  c.  c.  of  culture  diluted  slightly  the  strength  of  the  dis- 
infectant. 

Note  at  the  next  exercise  the  condition  of  each  inoculated 
tube.  From  them  the  approximate  strength  and  time  for 
the  disinfectant  to  destroy  the  bacteria  can  be  determined. 
When  this  is  found  the  more  exact  strength  and  time  can  be 
determined  by  repeating  the  experiment  with  weaker  dilu- 
tions or  shorter  exposures  or  both. 


EXERCISE  UV. 

TESTING  DISINFECTANTS  (continued). 

§  149.  General  directions.  Prepare  2  sets  of  tubes  con- 
taining 10  c.c.  each  with  2.5,  i.o  and  0.5%  solutions  of 
carbolic  acid  respectively,  and  test  their  effect  upon  the 
organisms  used  in  the  last  exercise  and  by  the  same  method. 
Note  the  conditions  of  the  inoculated  tubes  at  the  next 
exercise. 


85 

Examine  the  tubes  inoculated  during  the  preceding  ex- 
ercise. 

Allow  the  inoculated  tubes  to  remain  in  the  incubator  for 
several  days  and  note  whether  or  not  any  of  them  develop 
after  the  first  24  hours.  If  they  do  examine  them  micro- 
scopically to  determine  if  the  culture  is  pure.  Observe  in 
these  cultures  the  difference  between  immediate  destruction 
and  the  retarding  of  the  growth  of  the  bacteria. 


EXERCISE 

PASTEURIZING  AND  STERILIZING  MILK. 

§  150.  Explanatory  note.  Milk  is  pasteurized,  in  the 
present  acceptance  of  the  term,  when  all  of  the  pathogenic 
bacteria  which  it  may  happen  to  contain  (with  the  exception 
of  the  spores  of  Anthrax)  are  destroyed  with  the  more  im- 
portant Saprophytes.  It  is  not  necessarily  sterile  although 
it  sometimes  is.  The  temperature  and  time  for  heating  is 
from  60-68°  C.  for  20  minutes. 

In  this  exercise  it  is  the  purpose  to  study  the  effect  of  this 
process  on  the  bacteria  of  milk  and  to  compare  its  effect  with 
that  of  sterilization. 

§  151.  General  directions.  From  the  fresh  milk  pro- 
vided, make  3  agar  plates,  using  i,  2  and  3  loopfuls  respect- 
ively of  the  milk.  Put  20  c.c.  in  a  large  tube  and  set  it  in 
the  incubator.  Put  25  c.c.  in  each  of  4  large  test  tubes. 
Sterilize  2  of  them  by  boiling  for  30  min.  in  a  closed  water 
bath,  and  pasteurize  the  other  2  by  heating  them  in  the 
water  bath  for  30  minutes  at  65°  C.  It  requires  about  10 
minutes  for  the  milk  in  the  tubes  to  reach  the  temperature  of 
the  water,  leaving  the  milk  exposed  to  the  temperature  of 
the  water  for  20  minutes  and  cool. 

After  the  tubes  are  cooled  make  3  agar  plates  from  one  of 
the  tubes  treated  by  each  process,  using  i  loopful  of  milk  for 


86 

the  first  plate,  3  loopftils  for  the  second,  and  ^  c.c. 
(measure  with  a  graduated  pipette)  for  the  third.  Place  one 
of  the  tubes  of  milk  treated  by  each  process  with  the  plate 
cultures,  in  the  incubator,  and  leave  the  other  tubes  with  a 
tube  of  the  fresh  milk  at  the  room  temperature. 

At  the  next  exercise  note  carefully  the  condition  of  the 
milk  in  each  of  the  various  tubes,  and  also  the  number  of 
colonies  on  the  agar  plates. 

Keep  the  tubes  of  milk  for  further  examination  at  the 
following  exercise,  after  which  they  may  be  rejected. 


EXERCISE  LVL 

THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION  OF 

WATER. 

§  152.  Explanatory  note.  This  is  to  determine  the  num- 
ber of  bacteria  in  water.  In  preparing  media  for  this  pur- 
pose the  directions  given  in  the  Journal  of  American  Public 
Health  Association  for  Jan.,  1898,  p.  60,  should  be  followed. 
The  conditions  of  temperature  and  of  media  which  favor 
growth  differ  for  different  species.  Many  water  bacteria  will 
not  grow  at  the  incubator  temperature  while  others  which 
may  be  in  it  grow  very  slowly  at  the  room  temperature.  To 
determine  numbers  it  is  better  to  grow  the  bacteria  in  gelatin 
plates  at  the  temperature  of  the  room.  (In  an  actual  ex- 
amination a  much  larger  number  of  plate  cultures  should  be 
made  than  can  be  managed  here. ) 

§  153-  General  directions.  Make  from  the  properly  col- 
lected water  4  gelatin  plates  using  a  definite  quantity  of  wa- 
ter for  each.  It  may  be  safe  to  begin  with  to  inoculate  these 
tubes  with  o.  i,  0.25,  0.50,  and  i.oo  c.c.  respectively. 

To  determine  if  there  are  gas  producing  bacteria,  and  the 
approximate  number  of  these  if  any,  inoculate  10  fermentation 
tubes  with  i  c.  c.  each  and  5  with|  c.  c.  each.  (In  place  of 


87 

the  fermentation  tubes  glucose  agar  can  be  used.  In  this 
case  one  fermentation  tube  of  glucose  bouillon  should  be  in- 
oculated with  5  c.  c.  of  the  water  to  determine  the  quantity 
of  gas  produced  if  there  is  any).  From  the  gas  produced  in 
these  tubes  determine  approximately  the  number  of  the  gas 
producing  bacteria. 

Careful  and  full  notes  should  be  taken  on  this  examination 
the  preliminary  methods  for  making  a  bacteriological  exam- 
ination have  already  been  given  and  this  is  largely  in  the  na- 
ture of  an  investigation  by  each  student.  It  is  not  expected 
that  the  special  methods  other  than  those  used  in  the  lab- 
oratory for  pathogenic  bacteria  will  be  tried. 

§  154.  Collecting  water.  If  the  water  is  collected  from 
a  spicket  or  pump  allow  it  to  flow  for  2  or  3  minutes  first 
and  then  collect  the  desired  quantity,  100-200  c.  c.  in  a  ster- 
ile bottle  and  cork  tightly,  or  if  near  at  hand,  absorbent  cot- 
ton plugs  may  be  used. 

If  from  a  stream  or  river  withdraw  the  stopper  and  im- 
merse the  sterile  bottle,  to  the  depth  desired  and  allow7  it  to 
fill.  There  are  several  mechanical  devices  for  collecting 
water  from  considerable  depths  from  the  surface. 


EXERCISE  LVIL 

THE  QUANTITATIVE  EXAMINATION  OF  WATER  (continued). 

§155.  General  directions.  Examine  the  cultures  and 
count  the  colonies  on  the  plates  and  estimate  from  them  the 
number  of  bacteria  in  a  cubic  centimeter  of  the  water.  That 
is  if  there  are  40  colonies  on  the  plate  containing  .1  c.c.  of 
water  there  are  400  bacteria  in  i  c.c.  of  it. 

From  the  cultures  in  the  glucose  media  estimate  the  num- 
ber of  gas  producing  bacteria  present. 

Describe  the  appearance  of  the  different  colonies  and  indi- 
cate the  approximate  number  of  each  kind. 


88 

Keep  the  plate  cultures  until  the  following  exercise  and 
re-examine  and  count  the  colonies. 

Determine  the  obviously  different  genera  of  bacteria  by 
making  a  microscopic  examination  of  the  different  colonies. 

§  156.  Estimating  the  number  of  gas  producing  bac- 
teria in  water.  If  there  is  gas  in  all  of  the  ten  fermenta- 
tion tubes  inoculated  with  i  c.  c.  each,  it  would  show  that 
there  were  one  or  more  of  these  bacteria  in  each  cubic  centi- 
meter. If  3  of  the  5  tubes  inoculated  with  ^  c.  c.  each  con- 
tained gas  it  would  indicate  that  there  were  at  least  3  gas 
producing  bacteria  in  one  cubic  centimeter.  The  preliminary 
results  must  be  verified  by  repeated  examinations. 


EXERCISE  LVIIL 

THE  QUALITATIVE  EXAMINATION  OF  WATER. 

§  157.  Explanatory  note.  The  qualitative  examination 
of  water  consists  in  determining  the  species  of  bacteria 
present.  From  a  sanitary  standpoint  it '  consists  in  finding, 
if  present,  those  species  which  may  be  the  cause  of  disease 
among  people  or  animals  consuming  it.  The  pathogenic 
bacteria  which  may  be  in  the  water  will  depend  upon  the 
conditions,  but  usually  in  this  country  water  is  examined  for 
typhoid  and  hog  cholera  bacilli,  B.  coli  communis  and  B. 
pyocyaneus. 

In  India  the  spirillum  of  Asiatic  cholera  may  be  found  in 
the  water.  Occasionally,  anthrax  may  be  suspected.  It 
should  be  stated  that  B.  fluorescens  liquefaciens ,  pseudo  ty- 
phoid and  the  transitional  form  of  the  colon  group  are  to 
be  carefully  differentiated  from  B.  pyocyaneus  and  B.  ty- 
phosus.  Owing  to  imperfect  descriptions  many  of  the  com- 
mon soil  and  water  bacteria  cannot  be  readily  identified. 
The  genera  of  these  is  all  that  is  expected  here. 

§  158.  General  directions.  Make  at  least  four  gelatin 
plate  cultures,  and  such  others  as  may  seem  necessary  to 


89      y-/ 

determine  the  different  species.,  e^cjally  ^of  >  patik>geiiic 
bacteria,  and  their  relative  number  in  the  specimen  of  water 
furnished. 

Read  carefully  the  methods  for  water  analysis  in  the  text- 
books. 


EXERCISE  UX. 
THE  QUALITATIVE  EXAMINATION    OF  WATER    (continued). 

§  159.  General  directions.  Examine  the  cultures  made 
in  Exercise  LVIII,  and  make  such  others  (subcultures  from 
colonies,  etc.)  as  may  seem  necessary  to  determine  the  genera 
and  species  of  bacteria  present  with  the  relative  number  of 
each  per  cubic  centimeter. 


EXERCISE  LX. 

EXAMINATION   OF    CERTAIN   BACTERIA   NOT  STUDIED  IN 
THE   LABORATORY. 

§  1 60.  Explanatory  note.  This  exercise  will  be  devoted 
to  a  review  of  preparations  of  important  bacteria  not  studied 
in  the  laboratory  but  demonstrated  at  the  time  of  their  consid- 
eration in  the  lectures.  Unfortunately  the  number  neces- 
sarily omitted  is  large.  In  this  review  those  species  whose 
morphology  forms  an  important  part  in  their  identification 
such  as  the  bacillus  of  tetanus,  malignant  oedema,  and  vari- 
ous spirilla  will  be  considered.  Certain  of  the  pathogenic 
protozoa  will  also  be  demonstrated.  These  will  be  studied 
more  thoroughly  in  the  course  in  Pathology. 

§  161.  General  directions.  Examine  and  make  drawings 
of  the  bacteria  in  the  preparations  furnished. 

Complete  and  hand  in  all  notes  on  laboratory  work. 

Have  all  apparatus  for  individual  use  inspected  by  the  in- 
structor. 


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